Polypeptide-induced monoclonal receptors to protein ligands

ABSTRACT

The present invention relates to immunological receptors and ligands, and more particularly to monoclonal receptors raised to peptides whose amino acid residue sequences correspond to sequences of retroviral ligands. The receptors are used to assay body samples from a host to indicate exposure of the host to a carcinogen.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 925,815, filed Aug. 4,1992, abandoned, which is a continuation of application Ser. No.779,143, filed Oct. 21, 1991, abandoned, which is a continuation ofapplication Ser. No. 393,267, filed Aug. 12, 1989, abandoned, which is acontinuation-in-part of application Ser. No. 232,395 filed on Aug. 12,1988, abandoned, which is a continuation-in-part of application Ser. No.118,823 filed on Nov. 9, 1987, abandoned which is a continuation-in-partof Ser. No. 039,534 filed on Apr. 16, 1987, U.S. Pat. No. 5,015,571,that is a continuation-in-part of application Ser. No. 736,545 filed onMay 21, 1985, abandoned that is a continuation in part of applicationSer. No. 701,954, filed Feb. 15, 1985, U.S. Pat. No. 5,030,565 that is acontinuation in part of PCT application PCT/US84/01304 filed Aug. 17,1984 wherein the U.S. National Phase was entered on Feb. 15, 1985 Ser.No. 713,410, abandoned that is a continuation-in-part application ofU.S. application Ser. No. 524,084, filed Aug. 17, 1983, abandoned.

TECHNICAL FIELD

The present invention relates to immunological receptors and ligands,and more particularly to monoclonal receptors raised to polypeptides whowhose amino acid residue sequences correspond to sequences of retroviraloncoprotein ligands.

BACKGROUND

Retroviruses are viruses that contain a single strand of RNA as thegenetic material rather than DNA. The single-stranded RNA genome of eachof these viruses gives rise to a double-stranded DNA molecule after thevirus infects a susceptible host. This DNA replica of the viral genomethen introduces itself permanently into a chromosome of the successfullyinfected cell and replicates in that host chromosome.

The retroviruses discussed hereinafter and in the claims may be furtherdefined as being replication-defective retroviruses. Thus, these virusesdo not themselves contain a gene encoding the reverse transcriptaseusually required to permit the viral RNA genome to be translated into aDNA that can be introduced into a chromosome of the infected host.Rather, the retroviruses discussed hereinafter typically must becomplimented in their infection by a so-called helper virus that isreplication-competent. That second virus contains the gene that encodesthe reverse transcriptase enzyme that incorporates the genomic materialsfrom both viruses into the successfully infected host cells to transformthose cells.

For ease in understanding, the replication-defective retroviruses willbe discussed hereinafter and in the claims merely as retroviruses withthe understanding that they are replication-defective and require theassistance of a helper virus for successful infection and transformationof host cells. This usage of the term retrovirus is known in the art andhas been used in the art as such without further explanation.

Some members of the retrovirus family are highly oncogenic as judged bytheir ability to cause the formation of solid tumors within a shortperiod of time after being inoculated into the host. These viruses canalso cause "cancerous" changes in cells grown and cultured in thelaboratory; such changes are called "transformations" and provide areliable in vitro biological assay for oncogenic viruses. Several suchviruses have been isolated from chickens, turkeys, mice, rats, cats andmonkeys.

A single gene, the oncogene, located on the genome of these highlyoncogenic viruses is responsible for the tumorigenic potential of thevirus. In the case of several viruses, the protein products of theironcogenes, referred to herein as oncoproteins, have been immunologicallyidentified by taking advantage of the fact that serum from an animalbearing a virus-induced tumor contains antibodies directed against thoseoncoproteins.

A rapidly growing body of evidence indicates that the oncogenes ofretroviruses are closely related to and are derived from specificgenetic loci in the normal cellular genetic information of allvertebrates.

Interest in oncogenes has steadily risen in the last decade. AlthoughRNA tumor viruses have been implicated as the causative agents ofexperimentally induced neoplasia in chickens for over 50 years, it wasnot until the mid 1970s that mechanisms of virally induced neoplasiabegan to emerge Bishop (1983) Ann. Rev. Biochem. 52:301-54!. Accordingto one such mechanism, replication-competent arian viruses and defectivemammalian viruses had captured cellular genes that provided the viruseswith a transforming potential.

Molecular hybridization studies using specific nucleic acid probes,followed by genetic cloning of viral oncogenes and their cellularrelatives by recombinant DNA technology, have established the kinshipbetween retroviral oncogenes (v-onc) and cellular oncogenes (c-onc)found in all normal vertebrate cells. Molecular analysis of the severalretroviruses thus far isolated has revealed more than two dozendifferent oncogenes. In most cases, a corresponding cellular to theretroviral oncogene or oncoprotein has been isolated.

For example, the human EJ or T24 bladder carcinoma oncogene wasidentified as the homolog of the transforming gene of Harvey murinesarcoma virus (ras^(Ha)) and also of the BALB sarcoma virus (bas) Paradaet al., Nature, 297, 474-478 (1982); Der et al., Proc. Natl. Acad. SciUSA, 79, 3627-3634 (1982); and Santos et al., Nature, 298, 343-347(1982)!. In addition, the oncogene of the human carcinoma cell line LX-1was found to be homologous to the transforming gene of Kirsten strain ofmurine sarcoma virus (ras^(Ki)) Der et al., above!. Still further, thev-onc for a c-onc designated fps of avian origin is represented at leasttwice among a limited number of avian retrovirus isolates; its mammaliancognate designated fes in feline species is found in two differentstrains of feline sarcoma viruses.

The homology Doolittle et al., (1983) Science 221:275-277; Waterfield etal., (1983) Nature 304:35-39! between the gene product of the sisoncogene and one of the chains of platelet-derived growth factorprovided the most solid link between malignant transformation byoncogenes and stimulation of normal cell division by growth factors.This identity between oncogene products and growth factors and cellularreceptors was further substantiated with sequence analysis of theepidermal growth factor cellular receptor Downward et al., (1984) Nature307, 521-527; Ullrich et al., (1984) Nature 309:418-425! that was foundto be the normal homologue of erb B. Furthermore, immunologicalcross-reactivity of fms antibodies with colony stimulating factor-1receptor Sherr et al., (1985) Cell: 665-676! as well as protein kinasehomology with the insulin receptor Ullrich et al., (1985) Nature 313,756-761! and platelet derived growth factor receptor Yarden et al.,(1986) Nature 323; 226-232! indicated the kinase activity of many of thesequenced oncogenes would be important in the signal transduction ofseveral growth factors.

Sequencing of oncogenes captured by retroviruses or identified viatransfection experiments greatly extended the number of kinase familymembers. Hunter et al., (1985) Ann. Rev. Biochem. 54:897-930.! Thissequence analysis suggested the number of kinase-related proteins wouldbe large and the family members could be divided into subgroups basedupon sequence homology and overall structural similarities. The kinasefamily can be conveniently divided into gene products that do or do nothave extracellular (hormone/growth factor) binding domains.

The close similarity between the kinase portion of src and yes has beenapparent for several years. Kitamura et al., (1982) Nature 297:205-208.!Recently, sequencing of additional genes has extended this homology tofgr, Naharro et al., (1984) Science 222;63-66! lck, Marth et al., (1985)Cell 43:393-404. syn, Semba et al., (1986) Proc. Natl. Acad. Sci. USA83:5459-5463! and lyn Yamanashi et al., (1987) Mol. and Cell Biol.1:237-243!. All six of these genes encode proteins of approximately thesame size 55-65 kd, and the genes share intron/exon borders indicatingthey evolved from the same ancestral proto-oncogene. However, each geneis located on a separate chromosome and expresses different proteins indifferent tissues.

Many additional kinase family members can also be placed into subgroups.Mos Van Beveran et al., (1981) Nature 289:258-262! is closely related topim-1 Selten et al., (1986) Cell 46:603-611!, one of the preferredintegration sites of Moloney leukemia virus. Abl Reddy et al., (1983)Proc. Natl. Acad. Sci. USA 80:3623-3627! is closely related to arg Kruhet al., (1986) Science 234:1545-1547!. Fes Hampe et al., (1982) Cell30:775-785! and fps Shibuya et al., (1982) Cell 30:787-795!. representthe mammalian and avian counterparts of the same gene. Similarly, rafSutrave et al., (1984) Nature 309:85-88! and mil Mark et al., (1984)Science 224:285-289! are mammalian and avian homologues of the samegene. They are closely related to A-raf/pks Huleihel et al., (1986) Mol.and Cell Biol. 6:2655-2662; Mark et al., (1986) Proc. Natl. Acad. Sci.USA 83:6312-6316!.

A subgroup that does not have a viral counterpart contains genes thatencode protein kinase C, the receptor for phorbal esters. There are atleast three closely related genes comprising this subgroup Coussens etal., (1986) Science 233:859-866; Knopf et al., (1986) Cell 46:491-502!.Moreover, one of the genes can encode two proteins via alternative exonusage Ohno et al., (1987) Nature 325:161-166!. Other more distantlyrelated cytoplasmic kinases include cAMP- and cGMP-dependent proteinkinase Shoji et al., (1981) Proc. Natl. Acad. Sci USA 78:848-851; Takioet al., (1984) Biochemistry 23:4207-4218!, as well as myosin light chainkinase Takio et al., (1985) Biochemistry 24:6028-6037!. Severaltransmembrane kinases have also been sequenced in the past few years.

A gene closely related to the human epidermal growth factor receptor(HER) has also been found in humans (HER-2) Coussens et al. (1985)Science 230:1132-1139! and rats (neu) Bargmann et al., (1986) Nature319:226-230!. The growth factor that binds to ros Neckameyer et al.,(1985) J Virol. 53:879-884! is not known although the sequence is mostclosely related to the insulin receptor (HIR) Ullrich et al., (1985)Nature: 313, 756-761!. The colony stimulating factor 1 receptor, FMSHampe et al., (1984) Proc. Natl. Acad. Sci. USA 81:85-89!, forms asubgroup with kit Besmer et al., (1986) Nature 320:415-421! and thereceptor for platelet-derived growth factor, PDGF-R Yarden et al.,(1986) Nature 323:226-232!. In addition, sequences for the trkMartin-Zanca et al., (1986) Nature 319:743-748! and met-8 Dean et al.,(1985) Nature 318:385! oncogeneses have been published, although thecorresponding growth factors are not known.

A similar although not as extensive expansion has also been seen for thenucleotide binding proteins represented by the ras oncogene family.Sequence data indicate bas Reddy et al., (1985) J. Virol. 53:984-987! isthe mouse form of H-ras Dhar et al., (1982) Science 217:934-937!, andthat the H- and K-ras products differ principally at the carboxyl regionTsuchida et al., (1982) Science 217:937-939!. Through alternative exonsK-ras can encode 2 proteins (4A and 4B) McGrath et al., (1983) Nature310:501-506!. A third member, N-ras, also diverges from H- and K-ras inthis region Taparowsky et al., (1983) Cell 34:581-586!. Another closelyrelated gene is R-ras Lowe et al., (1987) Cell 48:137-146!, althoughthis gene is closely related to the three ras genes that have evolvedfrom the same ancestral gene, R-ras has different intron/exon boarder.Another gene, rho 7 Madule et al., (1985) Cell 41:31-40!, has scatteredregions of homology with ras. Furthermore, a third group, ral, also hassimilar regions of homology Chardin et al., (1986) EMBO J. 5:2203-2208!.Moreover, a yeast gene ypt Gallwitz et al., (1983) Nature 306:704-707!has regions of homology with ras and this gene is distinct from the twoyeast genes that have extensive homology with ras; i.e., they are morelike R-RAS.

Other genes that also have homology with ras include the G proteins Itohet al., (1986) Proc. Natl. Acad. Sci. USA 83:3776-3780! as well astransduction and elongation factor, Tu (Lochrie et al., (1985) Science228:96-99!. The G proteins are composed of subunits that stimulate(G_(s)) and inhibit (G_(i)) adenylate cyclase. Another related protein(G_(o)), has an unknown function. These proteins exists in a variety ofdifferent forms that have closely related sequences.

The nuclear proteins myb Rushlow et al., (1982) Science 216,1421-1423!,myc Colby et al., (1983) Nature 301:722-725! and fos van Straaten etal., (1983) Proc. Natl. Acad. Sci. USA 80:3183-3187! comprise anotherfamily of oncogenes that are related more by cellular location thansequence. However, additional genes related to these oncogenes have beenidentified. N-myc Stanton (1986) Proc. Natl. Acad. Sci. USA83:1772-1776! and L-myc Nau et al., (1985) Nature 318:69-73! sequenceshave been published, and unpublished related sequences have beenidentified. Moreover, the sequences are distantly related to fos. Arelated fos (r-fos) Cochran et al., (1984) Science 226:1080-1082!sequence has been published, and unpublished data indicate aphosphorylase inhibitor has limited homology as does the jun oncogene.

Another group of nuclear oncogene-related proteins include steroid andthyroid hormone receptors. Although only one sequence related to erb Ahas been published Sap et al., (1986) Nature 324:635-640; Weinberger etal., (1986) Nature 324:641-646!, hybridization studies indicate at leasttwo related sequences are present in the human genome Weinberger et al.,(1986) Nature 324:641-646!. Steroid receptor sequences indicate erb A(the thyroid hormone receptor) is part of a superfamily that includesseveral receptors (estrogen, glucocorticoid, progesterone, aldosterone)Greene et al., (1986) Science 231:1150-1153; Hollenberg et al., (1985)Nature 318:635-641; and Connelly et al., (1986) Science 233:767-770!.

In the growth factor group only the PDGF-1 chain Doolittle et al.,(1983) Science 221:275-277 and Waterfield et al., (1983) Nature304:35-39! has sequence homology to sis (PDGF-2). However, other growthfactors Gregory (1975) Nature 257:325-327; Marguardt et al., (1983)Proc. Natl. Acad. Sci. USA 80:4684-4688! (EGF and TGF) bind to theproduct of the erb B protooncogene, and CSF-1 Kawasaki et al., (1985)Science 230:291-296! binds to the fms protooncogene. Moreover, TGFDerynk et al., (1985) Nature 316:701-705!, forms another subgroup byvirtue of homologies with Mullerian inhibitory substance Cate et al.,(1986) Cell 45:685-698!, and the three chains that are found in thevarious forms of inhibition Mason et al., (1985) Nature 318:659-663 andVale et al., (1986) Nature 321:776-779!.

Finally, sequences representing two of the preferred integration sitesof MMTV have been published Van Ooyen et al., (1984) Cell 39:233-240 andMoore et al., (1986) EMBO J. 5:919-924!.

Thus, in the past few years, the number of related published sequenceshas increased dramatically. These sequences suggest that a limitednumber of pathways controlling cell division and differentiation existbut that many different members may participate in this control.

An example of transduction of only a portion of a cellular gene by aretrovirus is the erb B oncogene. The erb B oncogene is highlyhomologous to a portion of the ECG receptor Ullrich et al., Nature309:418 (1984)!, as already noted. Sequence analysis of the entirereceptor gene demonstrates the relatedness of erb B with the entireintracellular domain, the transmembrane domain, and a portion of theextracellular domain.

The protein encoded by the viral oncogene and the corresponding,homologous protein within the host cell are both referred to herein asoncoproteins, although the cellular oncoprotein is typically larger andis present in small quantities in normal cells, and thus need not onlybe associated with neo-plastic states. In addition, oncoproteins encodedby related oncogenes can have different molecular weights, e.g., the p85and pl08 oncoproteins encoded by v-fes^(ST) and v-fes^(GA),respectively, and the 100-105 kilodalton (also kd or K dalton) proteinof normal mink cells thought to be encoded by the c-fes gene. Sen etal., Proc. Natl Acad. Sci. USA, 80, 1246-1250 (1983).! The termoncoprotein is thus used generally herein for proteins whose genes andamino acid residue sequences are homologous, at least in part, asdiscussed hereinafter.

The oncoprotein is generally not present in the virus particle thatinfects the cell, but is only expressed after infection andtransformation. The corresponding cellular oncoprotein is expressed atmost minimally in normal cells and to a greater extent in neoplasticcells. Thus, the oncoprotein cannot typically be obtained from thevirus. In addition, isolation of oncoproteins from cells is madedifficult because of small amount present, the complex mixture ofproteins found in normal cells, and the relatively small amount of suchproteins present even in transformed cells.

Oncoproteins encoded by v-onc and c-onc genes thus typically containlarge sequences of amino acid residues that are homologous, butnevertheless are not usually identical. In addition, oncoproteinsencoded by genes of different viral strains, each of which containsostensibly the same oncogene, have been found to have slight variationsin their amino acid residue sequences as exemplified above, and by thefour published sequences of the ras gene which differ at the position ofthe twelfth amino acid residue. Thus, even when oncoproteins are inhand, it may be difficult to distinguish among them.

Immunologically induced receptor molecules such as monoclonal andpolyclonal antibodies or the idiotype-containing portions of thoseantibodies are useful in purifying protein ligands to which they bind,as diagnostic reagents for assaying the presence and quantity of theprotein ligands, as well as for distinguishing among homologous proteinligands.

The difficulties associated with obtaining quantities of oncoproteinstypically militate against the preparation of receptors to thoseoncoproteins, although whole cell-induced monoclonal antibodies to v-fesand v-fps encoded oncoprotein have been reported by Veronese et al., J.Virol., 43, 896-904 (1982). In addition, even were whole proteinsavailable for use as immunogens for inducing the production of suchreceptors, the use of large protein molecules as immunogens producesantisera containing polyclonal antibodies to the numerous epitopes ofthe large protein molecules.

Hybridoma and monoclonal antibody techniques utilizing whole proteins orlarge protein fragments as immunogens have been useful in narrowing theimmunological response to such immunogens. However, such technology asheretofore practiced has been extremely time consuming and has providedonly a relatively small number of hybridomas that secrete usefulantibodies that recognize the immunogen. Moreover, even when successful,such techniques cannot be predictive of the chemical identity of epitopeto which the receptor molecules are raised. Consequently, even afterimmunogen-recognizing receptors are produced, the obtaining of receptorsto specific, chemically identified epitopic portions of the proteinligand has been a hit or miss operation that still further reduces thenumber of useful hybridomas that are ultimately produced.

Arnheiter et al., Nature, 294, 278-280 (1981) reported on the productionof monoclonal antibodies that were raised to a polypeptide thatcontained 56 amino acid residues and corresponded in amino acid residuesequence to the carboxyterminal portion of an intact interferonmolecule. The 56-mer polypeptide thus corresponded to approximatelyone-third of the sequence of the intact molecule.

Arnheiter et al. reported on the production of eleven monoclonalantibodies. However, only one of those eleven monoclonal antibodiesbound both to the polypeptide immunogen and also to the intactinterferon molecule. In addition, that binding was not very strong asjudged by the 3000-fold excess of intact interferon required to competethe antibody away from the synthetic polypeptide. None of the othermonoclonal antibodies bound to the intact molecule.

In addition, the production of the hybridomas secreting those monoclonalantibodies required the spleens from three immunized mice. The low yieldof the desired interferon-binding monoclonal antibodies, and the factthat three mouse spleens were needed for the preparation of thosehybridoma cell lines indicates that those workers were relativelyunsuccessful in their efforts.

Lerner et al. have been successful in obtaining protection of animals bythe use of vaccines against pathogens by utilizing synthetic amino acidresidue sequences of short to moderate length as immunogens. SeeSutcliffe et al., Science, 219, 495-497 (1983).

However, it must be understood that until the present invention,successful preparation of hybridomas and their secreted monoclonalreceptors differs from the successful preparation of a vaccinecontaining oligoclonal receptors. Thus, for a high yield monoclonalantibody preparation, it is necessary to stimulate B-cells to secretelarge amounts of avid antibodies. On the other hand, for a syntheticvaccine, a wider spectrum of oligoclonal antibodies may be produced insmaller amounts and with lower avidities. In addition, protection of ananimal against a pathogen typically requires both T-cell and B-cellactivations so that a cellular response and a humoral response,respectively, can be induced in the animal.

A popular explanation for the success of syntheticpolypeptide-containing vaccines in generating antibodies that recognizeintact proteins and protect animal hosts involves a stochastic model inwhich the diversity of the immune response allows the observation of aninfrequent event; i.e., the polypeptide adopting the confirmation of itscorresponding sequence in the native molecule. The concept thatmoderate-length polypeptides can frequently conform to native structuresis contrary to theoretical and experimental studies. Rather, suchpolypeptides are thought to exist as an ensemble of a large number oftransient conformational states that are in dynamic equilibrium. T-Cellactivation by, and B-cell production of antibodies raised to, some ofthat conformational ensemble have been believed sufficient to provideprotection upon vaccination.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a monoclonal receptor molecule thatbinds both (a) to a protein ligand encoded by a retrovirus gene, and (b)to a polypeptide of moderate length, about 7 to about 40 residues, andpreferably about 10 to about 30 amino acid residues, having an aminoacid residue sequence corresponding to an amino acid residue sequence ofa portion of the protein encoded by a gene of a retrovirus. The receptormolecule is raised to (induced by) an immunogen containing thepolypeptide. Most preferably, the receptor molecule is a monoclonalreceptor of the IgG class of immunoglobulins.

Specific, preferred monoclonal receptor molecules of this invention bindto protein encoded by the oncogenes listed below, and also to thepolypeptide(s) listed opposite those oncogenes:

    ______________________________________                                        Oncogene  Polypeptide Sequence                                                ______________________________________                                        fes       SDVWSFGILLWETFSLGASPYPNLSNQQTR;                                               SPYPNLSNQQTR;                                                                 IGRGNFGEVFSG;                                                                 LMEQCWAYEPGQRPSF; and                                                         VPVKWTAPEALNYGR;                                                    myb       RRKVEQEGYPQESSKAG;                                                            RHYTDEDPEKEKRIKELEL; and                                                      LGEHHCTPSPPVDHG;                                                    fos       SGFNADYEASSRC;                                                                LSPEEEEKRRIRRERNIKMAAAKC; and                                                 RKGSSSNEPSSDSLSSPTLL;                                               sis       RKIEIVRKKPIFKKATV;                                                            RVTIRTVRVRRPPKGKHRKC; and                                           ras       YREQIKRVKDSDDVPMVLVGNKC;                                                      YTLVREIRQHKLRKLNPPDESGPGC;                                                    YTLVREIRQYRLKKISKEEKTPGC;                                                     KLVVVGARGVGK;                                                                 KLVVVGASGVGK; and                                                             KLVVVGAGGVGK;                                                       myc       CDEEENFYQQQQQSEL;                                                             PAPSEDIWKKFEL;                                                                LPTPPLSPSRRSGLC;                                                              CSTSSLYLQDLSAAASEC; and                                                       CTSPRSSDTEENVKRRT;                                                  mos       LPRELSPSVDSR;                                                                 IIQSCWEARGLQRPSA;                                                             LGSGGFGSVYKA;                                                                 RQASPPHIGGTY; and                                                             TTREVPYSGEPQ;                                                       erb-A     KSFFRRTIQKNLHPTYSC;                                                           VDFAKNLPMFSELPCEDQ; and                                                       CYGHFTKIITPAITRVVDFA;                                               erb-B     ENDTLVRKYADANAVCQ;                                                            LGSGAFGTIYKG; and                                                             IMVKCWMIDADSRPKF;                                                   PDGF-2    SLGSLTIAEPAMIAECK;                                                            RKIEIVRKKPIFKKATV; and                                                        RVTIRTVRVRRPPKGKHRKC;                                               PDGF-1    SIEEAVPAECKTR;                                                      EGF       CLHDGVCMYIEALDKYAC;                                                 abl       LMRACWQWNPSDRPSF;                                                             LGGGQYGEVYEG; and                                                             LWEIATYGMSPYPGIDLSQVY;                                              fms       FMQACWALEPTRRPTF; and                                                         LGTGAFGLVVEA                                                        src       LMCQCWRKDPEERPTF;                                                             LGQGCFGEVWMG; and                                                             CGSSKSKPKDPSQRRRS;                                                  yes       LMKLCWKKDPDERPTC; and                                                         LTELVTKGRVPYPGMVNREVL;                                              fgr       LTELTTKGRVPYPGMGNGEVL;                                              bas       KLVVVGAKGVGK;                                                       int-1     LHNNEAGRTTVFS;                                                      mil/raf   LVADCLKKVREERPLF; and                                                         IGSGSFGTVYRG;                                                       ros       LGSGAFGEVYEG;                                                                 VWETLTLGQQPYPGLSNIEVL; and                                                    LMTRCWAQDPHNRPTF.                                                   ______________________________________                                    

The present invention also contemplates a method of producing monoclonalreceptor molecules to a protein molecule ligand. In this method, animmunogenic polypeptide of moderate length (about 7 to about 40residues), preferably synthetically produced, or a conjugate of thatpolypeptide bound to a carrier is provided. The amino acid residuesequence of that polypeptide corresponds to a portion of the amino acidresidue sequence of a protein ligand. That immunogenic polypeptide, whenbound as a conjugate to a carrier of keyhole limpet hemocyanin and usedto immunize a mouse, is sufficiently immunogenic and antigenic toprovide a 50 percent binding titer of the immunized mouse's serum to thepolypeptide of at least about a 1:400 dilution after threeimmunizations, each containing at least 10 micrograms of polypeptide inthe conjugate and using complete Freund's adjuvant for the firstimmunization and alum as adjuvant in the second and third immunizations.

A mammal is hyperimmunized with the immunogenic polypeptide or aconjugate of that polypeptide bound to a carrier to provide ahyperimmune serum that exhibits a 50 percent binding titer to thepolypeptide of at least about a 1:400 dilution. The receptor moleculesof that serum also bind to the protein molecule ligand to which thepolypeptide corresponds in amino acid residue sequence.

The hyperimmunized mammal is maintained for a period of at least about30 days after the administration of the immunization that produces a 50percent binding titer of a dilution of at least about 1:400. A boosterimmunization, as by intravenous injection, is thereafter administered tothe animal.

Antibody-producing cells such as spleen cells (splenocytes) of theboosted mammal are fused with myeloma cells within a period of aboutthree to about five days from the day of booster administration toprepare hybridoma cells. The hybridoma cells so prepared are assayed forthe production of monoclonal receptor molecules that bind to a proteinmolecule ligand to a portion of which the immunogenic polypeptidecorresponds in amino acid residue sequence. Preferably, the hybridomacells are also assayed for the production of monoclonal receptormolecules that bind to the polypeptide.

The hybridoma cells that produce monoclonal receptor molecules that bindto the protein molecule ligand are then cultured to prepare anadditional quantity of such cells. In preferred practice, thosehybridoma cells that are cultured are also those that produce monoclonalreceptors that bind to the polypeptide.

Another embodiment of the present invention contemplates a diagnosticsystem such as a kit for assaying for the presence of an oncoproteinligand. This system includes at least a first package containingmonoclonal receptor molecules of this invention. Admixing apredetermined amount of those receptors with a predetermined amount ofan aqueous composition to be assayed for the presence of an oncoproteinligand forms a receptor-ligand complex by an immunological reaction whenthe oncoprotein ligand includes an amino acid residue sequencecorresponding to the amino acid residue sequence of the polypeptidebound by the receptor molecule. The presence of the complex can beidentified by a label that is preferably contained in a second packageof the system. A preferred oncoprotein ligand-containing aqueouscomposition includes a cell extract, amniotic fluid, urine, andconcentrated urine. The urine or urine concentrate is easily obtained bynoninvasive means and is readily concentrated to allow theimplementation of the diagnostic test set forth herein. Cell extractsand media conditioned by transformed cells are also suitable aqueouscompositions containing oncoprotein ligands.

An assay method is another contemplated embodiment of this invention.Here, a body sample to be assayed for the presence of an oncoproteinligand such as serum, a cell extract, amniotic fluid, urine or a urineconcentrate is admixed in a liquid solution containing anti-oncoproteinreceptor molecules. The admixture so formed is maintained for a periodof time sufficient for a complex (immunocomplex; reaction product orimmunoreactant) to form between an oncoprotein ligand and receptormolecule (antigen-antibody complex). The presence of a complex isthereafter determined.

Where urine, as obtained or in concentrated form, is the composition tobe assayed, anti-oncoprotein receptors of any origin, e.g., polyclonal,oligoclonal or monoclonal, can be used in the instant invention. Themonoclonal antibodies of this invention are utilized with other samplesto be assayed. Determinations of the presence of an immunoreactant aretypically carried out using a radioisotope- or enzyme-labeled antibodyor Staphylococcus aureus protein A that binds to the receptor of theformed immunocomplex.

A particularly novel aspect of this invention is the use of urine as abody sample. The assays described herein may be performed usingconcentrated urine as described, or may be performed using urine asobtained. Oncogene-related proteins have not been heretofore identifiedin urine samples.

The assay aspects of this invention can be conducted using a pluralityof oncoprotein-related polypeptide ligands to provide a pattern ofimmunological reactivity for a particular assayed sample. Patternsobtained are compared to patterns obtained from individuals having knowndisease states to provide a diagnosis.

A method for ascertaining the presence of a female fetus in utero isalso contemplated. Here, a sample of boiled, reduced, and preferablyconcentrated urine from a pregnant mother is admixed with receptormolecules that immunoreact with a polypeptide that has a formula,written from right to left and in the direction from amino-terminus tocarboxy-terminus, selected from the group consisting of: ##STR1## theurine sample being collected during the period about 16 through about 20weeks into the pregnancy. The admixture is maintained for a time periodsufficient for the receptor molecules to immunoreact with an oncoproteinligand present in the urine sample. The presence of a particularimmunoreactant is thereafter assayed for. The immunoreactant is thatformed between the receptor molecules and an oncoprotein ligand thatexhibits a relative molecular mass in a 5-17 percent polyacrylamide gelof about 40 kilodaltons for the receptor molecules that immunoreact withpolypeptide (i), above, and about 55 kilodaltons for the receptors thatimmunoreact with polypeptide (ii), above. The presence of animmunoreactant with either of those receptor molecules indicates thepresence of a female fetus in utero. The receptor molecules arepreferably monoclonal.

In yet another embodiment of the invention, monoclonal receptormolecules form the active, binding portions of an affinity-sorbantuseful for binding and purifying oncoprotein ligands. Here, thereceptors are linked to a solid support that is chemically inert to theoncoprotein such as agarose or cross-linked agarose. The affinitysorbant so prepared may then be admixed with an aqueous compositioncontaining a protein ligand to form a reversible receptor-ligand complexwhen the protein ligand has an amino acid residue sequence correspondingto the amino acid residue sequence of the polypeptide bound by thereceptor. The complex so formed can be thereafter dissociated to providethe protein ligand in a purified form.

The present invention provides several benefits and advantages.

One benefit of the invention is monoclonal receptor molecules that bindto epitopes contained in polypeptides of known amino acid residuesequence.

Another benefit of the invention is that monoclonal receptor moleculescan be raised that bind to epitopes contained in known amino acidresidue sequences of, oncoprotein ligands where those protein ligandsare not needed to induce the production of the receptor molecules.

One of the advantages of the present invention is the high yield methodof producing monoclonal receptors that bind to both an immunogenicpolypeptide of moderate length and to a protein ligand molecule to whoseamino acid residue sequence the polypeptide corresponds in part.

Another advantage of this invention is the provision of a diagnosticsystem such as a kit containing monoclonal receptor molecules capable ofassaying for the presence of an oncoprotein.

A further advantage of this invention is the provision of a diagnosticmethod that can be accomplished using body samples obtained bynon-invasive means.

Another advantage of this invention is that proteins of differingmolecular weights may be detected allowing a differential and highlyaccurate assessment of the precise oncogenes being expressed within theorganism.

A further advantage of this invention is the provision of a diagnosticmethod that allows prognostication of fetal development, or other growthstates including neoplasia that utilizes urine of the mother orindividual, respectively, in a non-invasive assay.

Still further benefits and advantages of the present invention will beapparent to those skilled in the art from the description and claimsthat follow.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a part of this disclosure:

FIG. 1 is a photograph of an autoradiograph illustrating animmunological assay for detecting the presence of the ST-FeSV v-fesoncoprotein. Cell extracts from approximately 10⁵ MSTF cells, aproductively transformed mink cell line infected with Snyder-Theilenstrain of feline sarcoma virus (ST-FeSV) and feline leukemia virus-B(FeLV-B) Sen et al., Proc. Natl. Acad. Sci. USA, 80,1246-1250 (1983)!,were electrophoresed onto a 5-17 percent polyacrylamide gel and thentransferred to nitrocellulose sheets. The transferred proteins were thenreacted with supernatants from hybridoma tissue cultures denominatedSl0F03 (lane 1) or S22C06 (lane 2) (ATCC designations ATCC HB 8596 andATCC HB 8595, respectively) or an anti-influenza hemagglutinin hybridomaused as a negative control. This procedure of polyacrylamide gelseparation followed by transfer to nitrocellulose and visualization isreferred to hereinafter as a Western blot procedure. Proteinvisualization was accomplished as described in the Materials and Methodssection, hereinafter.

FIG. 2 is a photograph of an autoradiograph illustrating animmunological assay for detecting the presence of the FeSV fusionprotein denominated p85 (85 kilodaltons; 85K daltons) by Western blotprocedures similar to those of FIG. 1. Cell extracts of approximately2×10⁶ MSTF cells were electrophoresed into a 5-17 percent polyacrylamidegel, and then electrophoretically transferred to nitrocellulose strips.The strips of nitrocellulose were incubated with 5 milliliters each ofhybridoma culture supernatant diluted 1:50 from hybridomas denominatedSl0F03 (lane A); P43D09 (lane B); P42Cl0 (lane C); P44E11 (lane D); orwith R₂ 06B08, an anti-Rauscher gp70 protein receptor-producinghybridoma Niman and Elder, Proc. Natl. Acad. Sci. USA, 77, 4524-4528(1980)!, as a negative control (lane E).

Binding was visualized by addition of peroxidase labeled rabbitanti-mouse IgG as is discussed in the Materials and Methods section,hereinafter. The marker "p85-" at the left side of FIG. 2 illustratesthe migration position of the 85k dalton ST-FeSV polyprotein encoded bythe fes gene.

As can be seen from the proteins in lane E, this technique permitsvisualization of protein molecules that are not specifically bound bythe monoclonal receptors of this invention. Subtraction of thenon-specifically bound proteins visualized in lane E from the proteinsvisualized in lanes A-D illustrates that the only specifically boundprotein is the p85 oncoprotein encoded by v-fes.

FIG. 3 is a photograph of an autoradiograph illustrating animmunoprecipitation assay for the presence of the ³² P-labeled FeSVfusion protein denominated p85. CCL64 mink cells (MSTF cells; lanes Band D) or those infected with FeLV-B and FeSV (MSTF cells; lanes A andC) were each labeled for 2 hours with 1 microcurie of ³² P. The labeledcell extracts were then incubated with 5 microliters of goat anti-FeLVpl5 antibodies (lanes A and B) or with 50 microliters of supernatantfrom cultured hybridoma Sl0F03 (lanes C and D). Immune complexes soprepared were collected using Staphylococcus aureus bacteria expressingprotein A. The precipitated complexes so collected were washed, and werethen dissociated into their component parts. The proteins werethereafter analyzed under reducing denaturing electrophoresis using a5-17 percent polyacrylamide gel. The markers "p8P-" and "pr65-" at theleft of FIG. 3 illustrate migration positions of the 85K dalton ST-FeSVfusion protein encoded by the fes gene, and the 65K dalton FeLVgag-precursor protein.

FIG. 4 is a graph illustrating immunoreactivities of oligoclonalantibodies raised to synthetic polypeptides corresponding in amino acidresidue sequence (i) to positions 139 through 155 of the predictedsequence of the simian sarcoma virus transforming protein denominatedp28^(sis) Devare et al., Proc. Natl. Acad. Sci. USA, 80, 731-735 (1983)!identified hereinafter as polypeptide (o) or number 113 and as PDGF2(73-89), and (ii) to residues 2 through 18 of the predicted amino acidresidue sequence of the avian myeloblastosis virus oncoprotein Rushlowet al., Science, 216, 1421-1423 (1982)! identified hereafter aspolypeptide (d) or number 131. The synthetic polypeptides conjugated tokeyhole limpet hemocyanin (KLH) were used to immunize mice as isdiscussed generally in the Materials and Methods section.

To test the specificity of oligoclonal antibody containing sera soprepared, 250 nanograms of unconjugated polypeptide or 500 nanograms ofKLH were dried onto the bottoms of microliter wells and fixed withmethanol as described by Niman and Elder, in Monoclonal Antibodies and TCell Products, Katz ed., CRC Press, Boca Raton, Fla., pp. 23-51 (1982).The remaining portions of the wells were blocked against non-specificprotein adsorption using 3% bovine serum albumin (BSA) and a 4 hourincubation period at 37 degrees C.

Into each well of the microliter plate was instilled 25 microliters eachof two-fold dilutions of immunized mouse sera, starting with a dilutionof 1:400, using tissue culture medium supplemented with 10% fetal calfserum and were incubated with the BSA-blocked polypeptide or KLH for 16hours a 25 degrees C. After washing 10 times with distilled water, 25microliters of rabbit anti-mouse kappa antibody (Libbon Bionics Inc.,Kensington, Md.) diluted 1:500 with 1% BSA in phosphate-buffered saline(PBS) were added and incubated for 2 hours at 37 degrees C. After anadditional 10 washings with distilled water, 25 microliters of goatanti-rabbit IgG conjugated to glucose oxidase and diluted 1:500 with 1%BSA in PBS were added and incubated for 1 hour at 37 degrees C.

The amount of glucose oxidase so bound was determined by addition of 50microliters of a solution containing 100 micrograms/milliliter of ABTSdye (Boehringer-Mannheim) in the presence of 1.2% glucose and 10micrograms/milliliter of horseradish peroxidase in 0.1 molar phosphatebuffer having a pH value of 6.0. The optical densities of the solutionsso prepared are read at 414 nanometers using a Titertech microscanner(Flow Laboratories Inc., Inglewood, Calif.).

Bindings exhibited by oligoclonal antibodies in sera raised to thesis-related and myd-related polypeptides are shown by open and closedsymbols, respectively. The antibody antigens are: sis-relatedpolypeptide (c) (, ◯); myb-related polypeptide (d) (▪, □); and KLH (♦,⋄).

FIG. 5 is a photograph of an autoradiograph illustrating animmunological assay for detecting the presence of non-reduced andreduced platelet-derived growth factor (PDGF) using mouse anti-seracontaining oligoclonal antibodies (receptors) induced by syntheticpolypeptides (c) and (d) as probes. PDGF extract was purified fromoutdated platelets as described in the Materials and Methods section.

Purified PDGF extract from approximately 2.5 units of platelets weremixed with a minimal volume of solution containing 0.5% sodium dodecylsulfate (SDS) and 5 percent of 2-mercaptoethanol. The resulting mixturewas boiled for 2 minutes and then electrophoresed therethrough a 5-17percent polyacrylamide gel. The protein was thereafterelectrophoretically transferred to nitrocellulose Niman and Elder,Virology, 123, 187-205 (1982)! that was thereafter cut into strips,following the Western blot procedure.

The nitrocellulose strips so prepared were then treated with a solutioncontaining 3% BSA, 0.1% polyoxyethylene (9) octyl phenyl ether (Triton®X-100, Rohm and Haas Company, Philadelphia, Pa.) in PBS to inhibitnon-specific protein binding. 4 Milliliters of mouse anti-serum diluted1:200 were then incubated with the nitrocellulose strips.

After washing 3 times with a solution of 0.1% Triton® X-100 in PBS, thenitrocellulose strips were incubated either with 10⁶ counts per minuteof ¹²⁵ I-labeled Staphylococcus aureus protein A (lanes 2 and 3), or a1:1000 dilution of peroxidase-conjugated goat anti-mouse serum (Tago,Inc. Burlingame, Calif.), and again washed with 0.1% Triton® X-100 inPBS. The peroxidase conjugate was developed with a solution containing0,009% H₂ O₂, 0.0025% 3,3'-dimethoxybenzidine dihydrochloride(Eastman-Kodak Co., Rochester, N.Y.) in a 10 millimolar Tris bufferhaving a pH value of 7.4. The ¹²⁵ I labeled strips were developed byexposure on XRP-1 film (Eastman-Kodak Co., Rochester, N.Y.) using CronexHi-Plus (E.I. DuPont de Nemours & Co., Wilmington, Del.) intensifyingscreens at minus 70 degrees C. for 48 hours.

Lane 1 contains the total protein stained with amido black. The purifiedplatelet extract is shown probed with anti-sera raised to thesis-related polypeptide (c) (lanes 2 and 4) or the myb-relatedpolypeptide (d) (lane 3 and 5) as a negative control. External molecularweight standards based on BSA, ovalbumin, chymotrypsinogen andbeta-lactoglobulin are shown on the left.

FIG. 6 is a photograph of an autoradiograph illustrating animmunological assay for the presence of PDGF following a Western blotprocedure similar to that described hereinbefore. PDGF was boiled in thepresence (lanes A-F) or absence (lanes G-L) of 10 percent2-mercaptoethanol prior to electrophoretic protein separation, followingthe procedures described in Niman, Nature, 307, 180-183 (1984). Twooligoclonal antibody-containing antisera induced by the amino-terminaltwelve amino acid residues of PDGF-1 denominated PDGF-1(1-12)! were usedin lanes A and G, and lanes B and H. Two oligoclonal antibody-containingantisera induced by a polypeptide from a central portion of PDGF-2denominated PDGF-2(73-89) and polypeptide (o)! that corresponds to theamino acid residue sequence at positions 139 through 155 of p28^(sis)were used in lanes D and J, and in lanes E and K. Oligoclonalantibody-containing antisera induced by the amino-terminal eighteenresidues of PDGF-2 denominated PDGF-2(1-18)! and by the twenty residuesof PDGF-2 located 36-16 residues from the carboxy terminus denominatedPDGF-2(126-145)!, corresponding to the sequence at positions 191 through210 of p28^(sis), were used in lanes C Mnd I, and lanes F and L,respectively. Antibody binding to the proteins was visualized usingrabbit anti-mouse IgG followed by 1.0⁶ cpm ¹²⁵ I-labeled Staphylococcusaureus protein A as described in Niman, supra, and in the Materials andMethods section hereinafter.

FIG. 7 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a 70,000 dalton protein in threecell lines using a Western blot procedure. An extract from approximately10⁶ cells per lane from each of SSV-transformed NIH 3T3 cells (lanesA-E), TRD1 cells (a spontaneously transformed Balb/3T3 cell line) (lanesF-J) and MSTF cells a mink lung line (CCL64) productively infected withFeLV-B and the Snyder-Theilen strain of FeSV! (lanes K-O) wastransferred to nitrocellulose sheets following a Western blot procedure.Oligoclonal antibody-containing antisera induced by PDGF-1(1-12) wereused in lanes A-C, F-H and K-M. Oligoclonal antibody-containing antiserainduced by PDGF-2(73-89) were used in lanes D,E,I,J,N and O. Theantisera were incubated with 100 micrograms of polypeptides PDGF-1(1-12)(lanes A,D,F,I,K and N), PDGF-2(1-18) (lanes B,G and L) andPDGF-2(73-89) (lanes C,E,H,J,M and O) prior to being immunoreacted withthe transferred cell extracts. Proteins were visualized as described forFIG. 6.

FIG. 8 is a photograph of an autoradiograph illustrating animmunological assay for the presence of p20^(sis) in culture mediaseparately conditioned by SSV-transformed normal rat kidney and normalrat kidney (NRK) cells.

Proteins from concentrated media, equivalent to 25 milliliters ofnon-concentrated media, conditioned by SSV transformed cells (lanesA,C,E and G) or NRK cells (lanes B,D,R and H) were separated andtransferred to nitrocellulose following the Western blot procedure. Thetransferred proteins were then admixed with oligoclonalantibody-containing antisera induced by PDGF-2(1-18) (lanes A-D) andPDGF-2(73-89) (lanes E-H). Sera were incubated with 100 micrograms ofpolypeptides PDGF-2(73-89) (lanes A,B,G and H) and PDGF-2(1-18) (lanesC,D,E and F) prior to being immunoreacted with the transferred proteins.Immunoreactions were visualized as described for FIG. 6. The marker"p20^(sis) " at the left side of FIG. 8 indicates The position ofp20^(sis).

FIG. 9 is a photograph of an autoradiograph illustrating animmunological assay for the presence of proteins encoded by or relatedto sis and fes antisera in urine from human cancer patients. The liquidbody sample in this assay was urine concentrate, obtained as describedin the Materials and Methods section. The concentrated urine waselectrophoresed into 5-17% polyacrylamide gel and then electrophoresedonto nitrocellulose.

Urine from three donors was concentrated 200-fold, dialyzed and 20microliters of each concentrate were electro-phoresed and the proteinstherein transferred to nitrocellulose as described before. These threedonors had a rectal tumor (lanes A,D,G and J), a liver tumor (lane B,E,Hand K) and a Ewing's sarcoma (lanes C,F,I and L). An oligoclonalreceptor-containing antiserum induced by the sis-related polypeptidePDGF-2(73-89) that had been preincubated with the immunizing polypeptidewas used in lanes D-F, while the same antiserum that had beenpreincubated with the fes-related polypeptide corresponding to thissequence located at positions 744-759 of the v-fes^(ST) oncoprotein wasused in lanes A-C. Similarly, an oligoclonal receptor-containingantiserum induced by the above fes-related polypeptide that had beenpreincubated with the immunizing polypeptide was used in lanes G-I,while the same antiserum that had been preincubated with the abovesis-related polypeptide was used in lanes J-L. Immunoreaction (binding)between the oligoclonal receptors and the proteins was visualized asdescribed for FIG. 6. The positions of the sis- and fes-related proteinsdetected in the urine concentrates are indicated on the left and rightmargins by the markers "sis" and "fes", respectively.

FIG. 10 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras-related proteins in urine.

Urine was concentrated 250-fold (lanes A and B), 35-fold (lane C),70-fold (lane D), 75-fold (lane E) and 325-fold (lane F). The urine wasdialyzed, 20 microliters of each concentrate were electrophoresed andthe proteins therein were transferred to nitrocellulose as describedbefore.

The donors had been diagnosed as normal (lanes A, B and F), or as havingone of the following conditions: 38 weeks pregnant (lane C), lymphoma(lane D) and colon carcinoma (lane E). The same normal patient providedthe urine samples that were collected 14 days apart and were used inlanes A, B and F.

All urine sample were assayed using 10 microliters of anti-ras ascitesfluid induced with residues 96-118 of the p21^(ras) (polypeptide 142)that had been preincubated with residues 744-759 of the polypeptidefes^(ST) (lane A); residues 96-118 of the polypeptide ras^(Ha) (lane B);or residues 138-154 of the polypeptide v-sis (lanes C-F). Immunoreaction(binding) between the oligoclonal receptors and the proteins wasvisualized as described for FIG. 6. The position of the ras-relatedproteins detected in the urine concentrates are indicated on the leftmargin by the marker "ras".

The protein detected that is related to the ras oncogene is detected bya monoclonal antibody secreted by the hybridoma denominated ATCC No. HB8679 that was raised to ras-related polypeptide 142. This protein ofapproximately 55K daltons was detected in lane A and the activity wasblocked by a preincubation with the immunizing peptide (lane B). Urinecollected from the same normal individual contained the same protein twoweeks later (lane F). This protein has been detected in the urine of apregnant patient (lane C) and of a cancer patient (lane D and E).

FIG. 11 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a 23K dalton protein in threecell lines using a Western blot procedure. The lanes of the Figure eachcontained an extract from about 10⁶ cells per lane from mink lung cellline transformed by the Snyder-Thielen strain of mink lung line sarcomavirus (MSTF) cells (lanes A-F) or from uninfected MSTF cell line CCL64(lanes G-L). The respective cell extracts were transferred frompolyacrylamide gel, onto nitrocellulose sheets, followed by a Westernblot procedure.

The extracts were assayed using antisera raised to polypeptide 142corresponding to residues 96-118 of p21^(ras) ("ras-1"; lanes A, B, G,H) that had been preincubated with polypeptide 141 corresponding toresidues 5-16 of v-ras^(HA) ("ras-2"; lanes A, G) or with polypeptide142 corresponding to residues 96-118 of p21^(ras) ("ras 1"; lanes B,H).

The same cell extracts were assayed with antisera raised to polypeptide121 corresponding to residues 519-530 of p85-fes ("fes-1"; lanesC,D,I,J) or to residues 744-759 of p85 fes ("fes-2; lanes E,F,K,L). Theantisera were preincubated with the fes-1 polypeptide (lanes D,J), withfes-2 polypeptide 744-759 (lanes F,L), or with the ras-1 polypeptide(lanes C,E,I,K) prior to being immunoreacted with the transferred cellextracts. Proteins were visualized as described for FIG. 6.

FIG. 12 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a secreted protein insupernatants from spontaneously transformed mouse 3T3 cell line TRD-1(lanes A,B) or a human T-24 bladder carcinoma line (lanes C,D). Thesupernatants were assayed for presence of secreted fes-related protein.

The cell lines were grown in the absence of serum and collected after 48hours of growth. 35 Microliters of 1500:1 concentration of T-24 cellline supernatant or 1000:1 concentration of TRD-1 cells wereelectrophoresed into a polyacrylamide gel, and then transferred ontonitrocellulose.

Mouse antisera to v-fes^(ST) synthetic polypeptide 127 corresponding toresidues 744-759 of p85^(fes) ("fes-2") were utilized for the assay. Theantisera were preincubated with synthetic polypeptide 121 correspondingto residues 519-530 of v-fes^(ST) ("fes-1"; lanes A and B), or with thefes-2 polypeptide used to raise the antisera (lanes B and D).

The antisera were then immunoreacted with the transferred cellsupernatant. Proteins were visualized as described for FIG. 6.

FIG. 13 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a ras related protein in a cellextract using a Western blot procedure.

A cell extract of approximately 10 spontaneously transformed mouse 3T3cells was used in lanes A-D. 35 Microliters of a 1500-fold concentrationof 48 hour supernatants from mouse 3T3 TRD-1 cells were used in lanesE-H. The proteins of the supernatants were electrophoresed in apolyacrylamide gel, and then transferred onto nitrocellulose.

Oligoclonal antibody-containing antisera to polypeptide 142corresponding to residues 98-118 of v-ras^(HA) were preincubated with anunrelated fes polypeptide (lanes A,C,E,G) or the ras polypeptide usedfor the immunizations (lanes B,D,F,H). Proteins were visualized asdescribed in FIG. 6.

FIG. 14 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras-, sis- or fes- relatedproteins in a cell extract using a Western blot procedure. The lanes ofthe Figure each contained an extract from about 10⁶ cells per lane ofmink lung cells transformed with the Snyder-Thielen strain of felinesarcoma virus (MSTF cells).

The extracts were assayed using antisera raised to polypeptidescorresponding to residues 96-118 of p21^(ras) (polypeptide 142, lane 2)to residues 1-18 of PDGF-2 (polypeptide 112, lane 1) And to residues744-759 of v-fes (polypeptide 127, lane 3). Proteins were visualized asdescribed for FIG. 6.

FIG. 15 is a photograph of an autoradiograph illustrating animmunological assay for the presence of a variety of proteins encoded byor related to sis, fes and ras oncogenes in urine using a Western blotprocedure similar to that described hereinbefore. The liquid body samplein this assay was urine concentrate, obtained as described in theMaterials and Methods section. The concentrated urine waselectrophoresed into 5-17% polyacrylamidize gel and then electrophoresedonto nitrocellulose.

Urine from 8 donors was concentrated 40-fold, dialyzed and 25microliters (the equivalent of 1 ml of unconcentrated urine) waselectrophoresed and the proteins therein transferred to nitrocelluloseas described before. These donors had multiple myeloma (lane 1, FIGS. 15and FIG. 15B), gastric cancer (lane 2, FIG. 15A and FIG. 15B; lane 1,FIG. 15C and FIG. 15D), 35 weeks pregnant (lane 3, FIG. 15A and FIG.15B), lymphoma (lane 4, FIG. 15A and FIG. 15B), gastric cancer (lane 1,FIG. 15C and FIG. 15D), 36 weeks pregnant (lane 2, FIG. 15C and FIG.15D), breast cancer (lane 3, FIGS. 15C and FIG. 15D) , 39 weeks pregnant(lane 4, FIG. 15C and FIG. 15D) and breast cancer (FIG. 15E).

Monoclonal or oligoclonal receptor-containing Antisera induced by sis-(FIG. 15A and FIG. 15B), ras- (FIG. 15C and FIG. 15D) or fes-relatedpolypeptides (FIG. 15E) were used to probe each sample to assay forimmunizing polypeptides. Twenty microliters of ascites fluid (induced byhybridoma ATCC HB 8679 and described hereinafter, and induced by ahybridoma raised to the sis related polypeptide 112 corresponding insequence to positions 1-18 of PDGF-2; FIGS. 15C and 15D, and 15A and15B, respectively) or mouse plasma (raised to a polypeptidecorresponding in sequence to positions 744-759 of the fes oncoprotein;FIG. 15E) were preincubated for 30 minutes at 37 degrees C. with 100micrograms of the immunizing ras polypeptide 142 (FIGS. 15A, 15D of FIG.15E), sis polypeptide 112 (FIGS. 15B and 15C) or fes polypeptide (FIG.15E, lane 3), with polypeptide 171 corresponding to positions 366-381encoded by erb B (FIG. 15E, lane 3), or with polypeptide 312corresponding to positions 590-605 of abl (FIG. 15E lane 4).

Following preincubation, the samples were diluted 1 to 1000 in 3 percentBSA, 0.1 percent Triton® X-100 in PBS at a pH value of 7.4. The antiserawere then assayed as described hereinabove. Binding was visualized asdescribed in FIG. 6.

FIG. 16 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras-, sis-, and fes-relatedproteins in urine.

Urine was collected at monthly intervals from a donor previouslydiagnosed as having active breast cancer (lanes 1, 4, 7, 2, 5, 8, 3, 6,9, FIG. 16A). Urine was concentrated and dialyzed and an equivalent of 1ml unconcentrated urine was applied to each lane of FIG. 16A.

In FIG. 16B, aliquots of the same sample used in Panel A, lanes 3, 6 or9 were applied at the following equivalents of unconcentrated urine;1000 microliters (lane 1); 500 microliters (lane 2); 250 microliters(lane 3); 125 microliters (lane 4); 60 microliters (lane 5); 30microliters (lane 6); 15 microliters (lane 7); 7.5 microliters (lane 8).

The samples were prepared and probed with oligoclonal antisera toras-(positions 96-118, polypeptide 142; FIG. 16A, lane 1-3; FIG. 16B),fes-(positions 744-759, polypeptide 127; FIG. 16A, lanes 4-6) orsis-polypeptide (PDGF-2 positions 1-18, polypeptide 112; FIG. 16A, lanes7-9) as described for FIG. 15 except that no preincubation Withsynthetic peptides was performed.

FIG. 17 is a photograph of an autoradiograph illustrating animmunological assay for the presence of ras- And fes-related proteins inurine. The donors of the assayed urine samples had been diagnosed ashaving recurrent breast cancer (lanes 1, 2) or were normal individuals(lanes 3-8).

The assay for ras-related proteins (FIG. 17A) and fes-related proteins(FIG. 17B) was conducted as described for FIG. 16. The samples assayedwere urine from a patient in clinical remission from breast cancer (lane1), the same patient 3 months later when the breast cancer reappeared(lane 2), and normal female (lanes 3-5), wherein samples were collected3 days apart, a normal female where samples were collected 12 hoursapart (lanes 6-7) and a normal male (lane 8).

FIG. 18 is a photograph of an autoradiograph showing the detection ofras-, fes- and sis-related proteins in urine samples from donors havingcancer. Urine from donors with bladder cancer (lane 1), prostate cancer(lane 2), prostate nodule (lane 3), or lymphoma (lane 4) were preparedand probed with antisera to sis (FIG. 18A), ras (FIG. 18B) or fes (FIG.18C) as described in FIG. 16. The bands migrating slightly slower thanp56^(s) is in lanes 1,2 represents excessive amounts of albumin in thesesamples. Although the increased levels of p56^(sis), p31^(sis), andp25^(sis), correlate with the increased albumin levels in FIG. 18A,lanes 1,2, other urine, samples from donors with bladder or prostatecancer contained increased levels of sis-related proteins in the absenceof elevated albumin levels (data not shown). The slowest migrating bandsin FIG. 18B, lanes 1-3 identify p100^(ras) while the bands slightlyfaster than light chain in FIG. 18B lanes 1-4 identify p21^(ras).

FIG. 19 is a photograph of an autoradiograph illustrating the detectionof oncogene-related proteins in urine from a pregnant donor.

Four urine samples from the same individual collected at one weekintervals during the final month of pregnancy were probed with antiserato sis-related polypeptide 112 (PDGF-2 position 1-18; FIG. 19A), raspolypeptide 142 (positions 96-118; FIG. 19B), or fes polypeptide 127(positions 744-759; FIG. 19C). Overexposure of FIG. 19C demonstrates thepresence of p35^(fes) lanes 3 and 4) and p40^(fes) (lane 4). The proteinmigrating slightly faster than the light chain band (FIG. 19C, lanes1-4) or at the bottom of the gel (FIG. 19C, lanes 2-4) was detected withthe mouse antisera to the fes peptide. In addition, a protein of 150,000daltons was also detected with the mouse antisera to the fes peptide.Urine samples were collected at one week intervals.

FIGS. 20, 21, and 22 are tables showing amino acid sequences of threeconserved regions of oncoproteins that have protein kinase activity.Those regions are denominated as "CONSERVED KINASE REGION" 1, 2 and 3,respectively, in FIGS. 20, 21 and 22. The oncogene encoding anoncoprotein having protein kinase activity is designated by its usualsymbol in the left-hand column. The middle column identifies thelocation in the oncoprotein polypeptide sequence, from theamino-terminus, of the conserved amino acid residue sequence. Theright-hand column shows the amino-acid residue sequences, from left toright and in the direction from amino-terminus to carboxy terminus, ofthose conserved regions. The amino acid residue sequences are also thesequence of polypeptides useful as immunogens for inducing production ofthe monoclonal receptors of this invention.

FIG. 23 is a table showing the frequency of detection ofoncogene-related proteins in urine samples of 51 control (normal donors)and 189 urine samples from donors with a variety of malignancies. Theamount of oncogene-related proteins in the urine was estimated usingimmunoblots, and placed into one of four categories: undetectable,detectable, 5- to 15-fold elevated and greater than 15-fold elevated.The remaining types are listed as composite.

p21^(ras) was detected in approximately 70 percent of all samples fromdonors having neoplastic tumor disease. However, similar frequencieswere found in apparently normal individuals. The most striking elevationof p21^(ras) was detected in samples from donors having ovarian andgastric cancer as well as myeloma and molar pregnancies, all of whichhad greater than 15-fold elevations of this protein in at least 30percent of the samples.

FIG. 24 is a table of data reflecting the detection of various levels ofthe oncogene-related proteins in 260 urine samples from pregnant donors.The samples were grouped according to the trimester of pregnancy.Multiple urine collections were obtained from many of the donors. Assayswere performed in accordance with the procedures and methods set fromhereinafter in the Materials and Methods section. As with the subset ofdonors having breast cancer, discussed hereinafter, very high levels ofp55^(ras) were detected in a group of pregnant donors throughout thecourse of pregnancy. sis- and fes-related proteins increased as thepregnancy proceeded.

The levels of p55^(ras) changed dramatically in the course of several ofthe pregnancies. In contrast, levels of p55^(ras) detected in multiplesamples from normal or breast cancer donors, the concentration ofras-related proteins increased greater than 15-fold in one week incertain donors.

The concentration of the three sis-related proteins was approximatelythe same throughout the last month of pregnancy. p35^(ras) was detectedin the final two weeks of pregnancy while p40^(ras) was detected only inthe final week.

Urine samples taken six weeks postpartum continued to contain elevatedconcentrations of these sis-related proteins although the ras- andfes-related proteins returned to normal (data not shown).

FIG. 25 shows an immunoblot of mink lung cells transformed by fes. Amink cell extract was probed with various antibodies to fes (lanes A-I)or erb B (lanes J,K).

FIG. 26 shows an immunoblot of human epidermoid carcinoma cells. Anextract of a human epidermoid carcinomal cell line was probed withantibodies as used in FIG. 1.

FIG. 27 shows an immunoblot of a concentrated urine sample from apregnant diabetic patient. A concentrated urine sample from a pregnantdiabetic patient was probed with antibodies used in FIG. 1.

FIG. 28 shows an immunoblot of an endometrial tumor extract. An extractof an endometrial tumor, (NIH Accession No. 071-781473-1), was probedwith antibodies to fes (lanes A-H) or erb B (lanes I-L). Antibodies inlanes A-D give reactivity patterns in ELISA assays different from thoseof lanes E-H. Lanes I and J are directed against domains of v-erb B, andlanes K and L produce a different reactivity pattern against the sameoncoprotein.

FIG. 29 shows an immunoblot of a breast tumor extract. An extract ofbreast tumor (NIH Accession No. 121-960-1), which metastasized to thelymph node was probed with antibodies used in FIG. 4.

FIG. 30 shows an immunoblot of breast tumor, (NIH Accession No.31-14459), an extract of which was probed with the antibodies used inFIG. 4.

FIG. 31 shows an immunoblot of ovarian tumor, (NIH Accession No.31-13530), an extract of which was probed with the antibodies used inFIG. 4.

FIG. 32 shows an immunoblot of a breast tumor, (NIH Accession No.121-960-1), which metastasized to a lymph node. This metastic breasttumor extract was probed with antibodies to ros (lane A), fes (lane B),β TGF, (lanes C-G), ras (lanes H-J), and erb B (lanes K-N).

FIG. 33 shows an immunoblot of a metastatic ovarian carcinoma extractderived from NIH tumor 31-18265. This carcinoma metastasized to theomentum and was probed with the antibodies used in FIG. 8.

FIG. 34 shows an immunoblot of a metastatic colon carcinoma (NIHAccession No. 31-18152) which metastasized to the lymph node. Thisextract was probed with the antibodies used in FIG. 8.

FIG. 35 shows an immunoblot of a metastatic ovarian carcinoma (NIHAccession No. 031-10128-1). This extract of an ovarian carcinoma whichmetastasized to the omentum was probed with the antibodies used in FIG.8.

FIG. 36 shows an immunoblot of a lymphoma (NIH Accession No.021-50073-1) from the spleen. This extract was probed with theantibodies used in FIG. 8.

FIG. 37 shows an immunoblot of a breast carcinoma extract (NIH AccessionNo. 031-1239-1). This extract was probed with the antibodies used inFIG. 8.

FIG. 38 shows an immunoblot of a rectal tumor extract (NIH Accession No.31-19066). This extract was probed with the antibodies used in FIG. 8.

FIG. 39 shows an immunoblot of a metastic lung carcinoma, (NIH AccessionNo. 041-78297-1). An extract of this lung carcinoma which metastasizedto a lymph node was probed with the antibodies used in FIG. 8.

FIG. 40 shows an immunoblot of rat striatum. An extract of rat striatumtaken from 18 day old embryo, and 2 day old, 18 day old, 70 day old, and1 year old rats were probed with H/N-RAS (lane A), H-RAS (lane B), MYC(lane C), v-myb (lane D), int-1 (lane E), and two different SIS directedantibodies (lanes F and G).

FIGS. 41 and 42 are tables showing reactivity patterns of tumor extractsderived from cell lines on deposit at the NIH depository. The extractswere probed with various antibodies, and the resultant patterns werescored for the presence and level of oncogene product. The scoring wasbased on band intensities derived using the immunoblot technique.

FIG. 43 shows the asynchronous appearance of oncogene-related protein inthe urine of a gestational trophoblast disease patient undergoingchemotherapy. Sequential urine samples from a gestational trophoblastdisease patient undergoing chemotherapy were probed with antibodiesdirected against SIS residues (lane A) H/N-RAS residues (lane C), MYCresidues (lanes D and E), src residues (lane F and G), and int-1residues (lane H).

FIG. 44(A). Immunoblot of serum of normal, healthy control. The sampleof 100 μl of serum was probed with antibodies to peptide sequencespredicted by the following oncogenes: sis (lane 1), fes (lanes 2, 3 and15), β-TGF (lanes 4 and 5), int-1 (lanes 6 and 12), myb (lane 7), src(lane 8), c myC (lanes 9, 13 and 14), mos (lane 10), and H-ras (lanes 11and 16).

FIG. 44(B). Immunoblot of serum of individual with multiple carcinogenexposure (PCB's, asbestos, cigarette smoke) but without clinicallydetectable malignant disease. The sample of 100 μl of serum was probedwith antibodies to peptide sequences predicted by oncogenes as definedin FIG. 1(A). Note the presence of prominent lower bands in lanes 11 and16 not found in the normal, healthy control; these bands correspond tothe Harvey ras oncogene-encoded P21 protein.

FIG. 45. Immunoblot of serum of patient with multiple exposures tocarcinogens, (a) 18 months prior to clinical manifestation of thecolonic polyp, and (b) 6 weeks after removal of the polyp. Note thepresence of the band for the ras encoded p21 protein in (a) which is notseen in (b).

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates monoclonal receptor molecules tooncoprotein ligands, a general method of inducing or raising suchreceptors, and products and methods that utilize those receptors. Termsused frequently herein are defined as follows:

Receptor--A "receptor" is a biologically active molecule that binds to aligand. The receptor molecules of this invention are intact orsubstantially intact antibodies or idiotype-containing polyamideportions of antibodies. Biological activity of a receptor molecule isevidenced by the binding of the receptor to its antigenic ligand upontheir admixture in an aqueous medium, at least at physiological pHvalues and ionic strengths. Preferably, the receptors also bind to theantigenic ligand within a pH value range of about 5 to about 9, and ationic strengths such as that of distilled water to that of about onemolar sodium chloride.

Idiotype-containing polypeptide portions (antibody combining sites) ofantibodies are those portions of antibody molecules that include theidiotype and bind to the ligand, and include the Fab and F(ab')₂portions of the antibodies are well known in the art, and are preparedby the reaction of papain and pepsin, respectively, on substantiallyintact antibodies by methods that are well known. See for example, U.S.Pat. No. 4,342,566 to Theofilopolous and Dixon. Intact antibodies arepreferred, and will be utilized as illustrative of the receptormolecules contemplated by this invention.

Monoclonal receptor--A "monoclonal receptor" (Mab) is a receptorproduced by clones of a single cell called a hybridoma that secretes butone kind of receptor molecule. The hybridoma cell is fused from anantibody-producing cell and a myeloma or other self-perpetuating cellline. Such receptors were first described by Kohler, and Milstein,Nature, 256, 495-497 (1975), which description is incorporated byreference.

Oligoclonal receptor--An "oligoclonal receptor" is a receptor that isinduced by and binds to more than one epitope on a polypeptide ofmoderate length such as about 7 to about 40 or more preferably about 10to about 30 amino acid residues long. Oligoclonal receptors are usuallya mixture of receptors produced by more than one cell. Oligoclonalreceptors so produced are usually more epitopically specific in theirbinding than are the polyclonal receptors raised to whole proteinmolecules that can have epitopic regions throughout the length of theprotein chain or chains. Animals immunized with the polypeptides usefulherein produce sera containing oligoclonal receptors (antibodies).

Ligand--A "ligand" is the protein or polypeptide to which a receptor ofthis invention binds.

Corresponds--The term "corresponds" as used herein in conjunction withamino acid residue sequences means that the amino acid residue sequenceof a first polypeptide or protein is sufficiently similar to the aminoacid residue sequence contained in a second polypeptide or protein sothat receptors raised to the first (e.g., an antigenic syntheticpolypeptide) immunologically bind to the second (e.g., an oncoprotein)when the two are admixed in an aqueous composition. Such correspondingpolypeptides and/or proteins can also be said to contain homologousepitopes, and therefore share homologous sequences of at least about 6to about 8, e.g., 7, residues.

The epitope-containing amino acid residue sequences of the correspondingfirst and second polypeptides or proteins are most preferably identical.However, changes, preferably conservative, in amino acid residues, anddeletions or additions or residues, within the epitope may be made andstill permit the cross-reaction of a receptor to the first polypeptideor protein with the second, as is known. Conservative changes in aminoacid residues are well known, and include exchanges of residues betweenlysine (Lys; K) and arginine (Arg; R), between aspartic acid (Asp; D)and glutamic acid (Glu; E), between leucine (Leu; L) and isoleucine(Ile; I) and the like.

The preferred polypeptides useful herein are frequently described ashaving an amino acid residue sequence that corresponds to a portion ofamino acid residue sequence of a protein. Such polypeptides preferablyonly contain amino acid residue that correspond identically, in additionto terminal residues such as Cys residues utilized for binding orlinking the polypeptides to a carrier. Additional amino acid residuesthat do not correspond to residues in the protein may also be present atpolypeptide termini, but the use of such residues, while contemplatedherein, is usually wasteful, and is not preferred.

Similarly, proteins are described as having an amino acid residuesequence to a portion of which the amino acid residue sequence of apolypeptide corresponds. This terminology is intended to imply the samerelationship between the polypeptide and protein discussed hereinabove.

The full names for individual amino acid residues are sometimes usedherein as are the well-known three-letter abbreviations. The one-lettersymbols for amino acid residues are used most often. The Table ofCorrespondence, below, provides the full name as well as theabbreviations and symbols for each amino acid residue named herein.

    ______________________________________                                        Table of Correspondence                                                                         Three-letter                                                                            One-letter                                        Amino acid        abbreviation                                                                            symbol                                            ______________________________________                                        Alanine           Ala       A                                                 Arginine          Arg       R                                                 Asparagine        Asn       N                                                 Aspartic acid     Asp       D                                                 Asparagine + aspartic acid                                                                      Asx       B                                                 Cysteine          Cys       C                                                 Glutamine         Gln       Q                                                 Glutamic acid     Glu       E                                                 Glutamine + glutamic acid                                                                       Glx       Z                                                 Glycine           Gly       G                                                 Histidine         His       H                                                 Isoleucine        Ile       I                                                 Leucine           Leu       L                                                 Lysine            Lys       K                                                 Methionine        Met       M                                                 Phenylalanine     Phe       F                                                 Proline           Pro       P                                                 Serine            Ser       S                                                 Threonine         Thr       T                                                 Tryptophan        Trp       W                                                 Tyrosine          Tyr       Y                                                 Valine            Val       V                                                 ______________________________________                                         (A. L. Lehninger, Biochemistry; Worth Publishers, Inc., N.Y., N.Y., 1970)

I. PRODUCTION OF MONOCLONAL RECEPTORS

As noted previously, the present invention contemplates monoclonalreceptor molecules that bind to an immunogenic polypeptide of moderatelength, e.g., about 7 to about 40 residues and preferably about 10 toabout 30 residues, as well as binding to a protein molecule ligand, aportion of whose amino acid residue sequence corresponds to the aminoacid residue sequence of that polypeptide. The monoclonal receptors ofthis invention are raised or induced by use of an immunogenicpolypeptide or conjugate of that polypeptide linked to a carrier; theimmunogenic polypeptide containing an amino acid residue sequence ofmoderate length corresponding to a protein of the amino acid residuesequence of the protein molecule ligand.

Epitopic localization of monoclonal antibodies poses technical problems.Monoclonal antibodies to the entire bacterial gene products can beproduced with two different types of immunogens, native or denatured.Use of native protein poses the most serious technical problemsregarding purification and subsequent epitope mapping. The chiefadvantage of using a native protein is the production of monoclonalantibodies that block the biological function of the target protein.

The oncogene product produced in bacteria is typically not structurallythe same as the gene product synthesized in higher organisms. Directevidence for this difference is provided by analysis of the sis geneproduct. In mammalian cells the p28^(sis) is rapidly cleaved intop20^(sis). In contrast, bacterial p28^(sis) is not cleaved nor does itform a dimer.

Indirect evidence for differences between other oncogene productsproduced in bacteria or avian cells is provided by the observation thatmonoclonal antibodies raised against the E. coli-produced proteinproduct bind much more efficiently to the immunogen than to the proteinsynthesized in transformed chicken cells, even though the immunogen wasdenatured.

It is seen that the sequence of the viral oncogene can provide a basisfor identifying additional regions of a protooncogene sequence that canbe useful for synthesizing additional peptides for the generation andisolation of additional monoclonal receptors. Similarly, the sequenceanalysis of these proto-oncogenes identifies additional related peptidesthat have not yet been isolated in a retrovirus.

Thus, although purification of denatured protein is technically easier,the resulting antisera may recognize conformations unique to thebacterial gene product. This observation poses serious technicaldifficulties for epitope mapping studies.

Approaches for defining the epitope of the antibodies employ proteinfragments generated by partial proteolysis or expression if subgenomicfragments. Although mapping of epitopes using protein fragments wasfirst demonstrated by Niman and Elder, Proc. Natl. Acad. Sci. USA, 77,4524 (1980), only an approximation of the binding sites could be madeeven when several digests were assayed with a large panel of monoclonalantibodies. Thus, immunization even with protein fragments limits thedefinition of the binding site. Furthermore, there is no guarantee thatregions of interest will induce monoclonal antibodies.

In contrast, immunization with appropriate polypeptides of known aminoacid residue sequence as carried out herein, assures a production ofantibodies (receptors) that immunoreact with well defined regions; i.e.,regions that correspond to the sequences of the immunizing polypeptides.

Mapping of epitopes suggests that changing the epitope by one amino acidmay produce markedly different reactivities, while other studies showthat cross-reactivities are obtained when one or more amino acidresidues are different within the epitope. Furthermore, immunization ofthe same strain of mouse with the same synthetic polypeptide may producedifferent reactivities detected in the serum.

Hybridomas produced with synthetic polypeptides also produce monoclonalreceptors that react with the intact protein under a variety of reactionconditions because the recognition is largely conformationallyindependent. Therefore, Western blot, dot blot, fixed cells, and fixedtissues and body fluids such as cellular extracts, amniotic fluid, andurine, either concentrated or as obtained, can be assayed a well asnative proteins. Furthermore, the known, precisely defined amino acidresidues in the epitope allow isolation of antibodies that candistinguish single amino acid changes, therefore providing a means ofdetermining the significance of limited changes in conserved regions ofrelated proteins.

Monoclonal antibodies against synthetic polypeptides also provide ameans of mapping sites of protein interaction. Differentialcoprecipitations of molecules associated with pp60^(src) have beenreported, suggesting identification of regions of src proteins that areinvolved in such interactions.

Thus, inducing the production of monoclonal antibodies (receptors) withan immunogenic synthetic immunoreact with domains defined by thesequence of the immunizing polypeptide does not require complexmethodologies for isolation of isolation of the correspondingimmunogenic oncoprotein or the identification of that oncoprotein'sepitopic site, and produces receptors that recognize the oncogeneproduct in a conformation independent manner, all of which broaden theapplication of such receptors for a variety of studies.

It was noted previously that although animal host protection has beenshown to be possible by the use of immunogenic polypeptides as theactive agents in vaccines, the ability to utilize such immunogenicpolypeptides to produce high yields of hybridoma antibodies (Mabs) wasnot heretofore thought a likely possibility. Since each Mab is derivedfrom a single cell that produces only one specificity, the ratio of thenumber of clones producing anti-polypeptide antibodies that alsorecognize the intact protein molecule, to the total number ofpolypeptide recognizing clones can provide a reasonable estimate of thetrue confirmational frequency of the polypeptide.

The results described herein are contrary to the before-mentionedstochastic model, and the frequency for the moderate-length polypeptidesused herein assuming a conformation similar to that of the nativeprotein is much higher than was previously expected. The frequency ofproducing hybridomas whose Mabs recognize both the synthetic polypeptideto which they were raised and the intact molecule is about 4 orders ofmagnitude (about 10,000) times greater than that predicted by thestochastic theory.

It is also noted that various workers have been utilizing immunogenicpolypeptides to raise antibodies that recognize those polypeptides forseveral decades. In addition, the above-referenced Kohler and Milsteinarticle as to the production of monoclonal antibodies was published in1975. Since that date, 1975, Arnheiter et al., Nature (London), 294,278-280 (1981) described an attempt to prepare a monoclonal antibodyusing a polypeptide immunogen. As was previously noted, the Arnheiter etal. results must be viewed as a failure in that those authors requiredthe use of the spleens of three immunized mice and obtained only one IgGtype monoclonal antibody that recognized their large, 56-mer polypeptideas well as the protein to whose sequence that polypeptide corresponded.

It is believed that the relative paucity of published reports relatingto the preparation of monoclonal receptors prepared from immunogenicpolypeptides that recognize both the immunogen and a protein ligand towhose amino acid sequence the immunogenic polypeptide corresponds inpart is due to at least two factors. First, the prevalent thoughtfollowing the stochastic model predicts that few if any such monoclonalantibodies could be prepared. Second, the fact that workers such asArnheiter et al., above, did not possess a method suitable for thepreparation of the monoclonal receptors, inasmuch as the monoclonalreceptors of this invention that are raised to polypeptides are prepareddifferently from monoclonal antibodies prepared to whole proteins.

Thus, to successfully prepare IgG class monoclonal receptors thatrecognize both the immunogenic polypeptide and the protein ligand towhose amino acid residue sequence that polypeptide corresponds in part,one should follow the steps outlined hereinbelow.

An immunogenic polypeptide alone, or as a conjugate of that polypeptidebound (linked) to a carrier is provided. The polypeptide has an aminoacid residue sequence of moderate length, such as about 7 to about 40amino acid residues, and preferably about 10 to about 30 residues. Theamino acid residue sequence of the immunogenic polypeptide correspondsto a portion of the amino acid residue sequence of a protein moleculeligand such as an oncoprotein. While the immunogenic polypeptide can beused by itself as a ligand, it is preferred to use the polypeptideimmunogen as a conjugate bound to a carrier such as keyhole limpethemocyanin (KLH), albumins such as bovine serum albumin (BSA), humanserum albumin (HSA), red blood cells such as sheep erythrocytes, tetanustoxoid and edestin, as well as polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.

The immunogenicity and antigenicity of the polypeptide may be tested bybinding the polypeptide to a keyhole limpet hemocyanin carrier as aconjugate, and then using the conjugate so prepared to immunize a mouse.The immunizing polypeptide or conjugate is dissolved or dispersed in aphysiologically tolerable diluent such as normal saline,phosphate-buffered saline or the like as are well known in the art. Anadjuvant, discussed below, is also included in the inoculum used forimmunizations.

A useful polypeptide is sufficiently immunogenic and antigenic toproduce a 50 percent binding titer of the immunized mouse's oligoclonalreceptor-containing anti-serum to the polypeptide that is at least abouta 1:400 dilution after three immunizations in a one-month period, eachof which immunizations contains at least about ten micrograms, andpreferably at least about 50 micrograms, of the polypeptide in theconjugate, and utilizing complete Freund's adjuvant for the firstimmunization and alum as adjuvant thereafter.

This test procedure need not be carried out prior to the use of a givenpolypeptide as immunogen, but it is preferable to do so as apre-screening technique to determine that polypeptides will be useful inpreparing the desired monoclonal receptors. Whether used as a pre-screenor not, the polypeptides useful herein as immunogens provide the abovetiter using the above immunization regimen.

Upon provision of the immunogenic polypeptide, a mammal such as a mouse,rabbit, goat, horse or the like, is hyperimmunized with the immunogenicpolypeptide or conjugate of that polypeptide bound to a carrier toprovide a hyperimmune serum whose receptor molecules exhibit a 50percent binding titer to the polypeptide of at least about a 1:400dilution. Thus, the same animal, e.g., a mouse, in which one may desireto pre-test the immunogenicity of the polypeptide may be used forraising the Mabs.

It is particularly preferred that the same animal that is used for apre-test be used for raising the Mabs. This preference stems from thefact that once the above 50 percent binding titer is achieved, thepreparation of hybridomas secreting monoclonal antibodies of the desiredspecificity using the spleen of that animal as the source ofantibody-producing cells is substantially assured, aside from theoccurrence of random laboratory mishaps such as contamination of cellcultures or otherwise destroying those cultures.

It is noted that the immunization regimen required to provide ahyperimmune state is a function, inter alia, of the animal type, animalweight, the immunogenicity and amounts of the polypeptide and carrier,if used, the adjuvant, if used the number of immunizations administeredin a given time period, as is known. The above-described regimen forobtaining a 50 percent binding titer dilution of at least about 1:400provides a hyperimmune state in the test mouse and may be used as aproportionalizable basis for inducing hyperimmune states in otheranimals. It is further noted that three immunizations are notnecessarily required to provide the hyperimmunized state, but for auseful polypeptide, three such immunizations in a one-month period aresufficient to produce that state, or the polypeptide is not sufficientlyimmunogenic for the high yield production of hybridomas and theirmonoclonal antibodies of this invention.

The serum oligoclonal receptor molecules so produced in thehyperimmunized animal also bind to the protein molecule ligand, to aportion of which the immunogenic polypeptide corresponds in amino acidresidue sequence. Binding assays are described in the Materials andMethods Section hereinafter. It is noted that a pure sample of theprotein molecule ligand need not be utilized in these assays but rather,a cell extract or tissue preparation such as a microscope slidecontaining the protein ligand may be utilized.

The hyperimmunized animal is maintained; i.e., kept alive withoutadministration of further immunizations for a period of at least about30 days after administration of the immunization that produces a 50percent binding titer of at least a 1:400 dilution. In other words, theanimal is first immunized to provide a hyperimmunized state, and thenthe hyperimmunization allowed to recede.

The decline in binding activity typically takes one to about fivesmonths for mice. This decline in binding titer is believed to correspondto a period in which primed blast cells become capable of mounting avigorous response when the immunogen is again introduced.

A booster immunization, as by intravenous injection, using theimmunogenic polypeptide or its conjugate is administered to the animalafter the period of maintenance is completed, e.g., at least 30 daysafter the last immunization. Antibody-producing cells, such as spleencells or lymph cells of the boosted animal are then fused with a myelomacell from the same animal type (species) within a period of about threeto about five days from the day of booster administration to preparehybridoma cells. The boost is believed to stimulate the maturation ofthe blast cells to the point at which those cells secrete nearly optimalamounts of oligoclonal antibodies to the polypeptide.

The SP2/O-Agl4 (ATCC CRL 1581), hypoxanthine-aminopterin-thymidine(HAT)-sensitive, myeloma cell line is preferred for use in fusion withmouse spleen cells, although other cell lines such as P3X63-Ag8.653 mayalso be utilized. Details using this HAT line for fusion are givenhereinafter in the Materials and Methods Section. The hybridoma cellsare thereafter cloned at limiting dilution free from the presence of, orneed for, feeder layers of macrophages to reduce overgrowth bynon-producing cells, and to provide a selection method for cells whichgrow readily under in vitro conditions. Such feeder layers may, however,be used.

The hybridoma cells so prepared are then assayed for the production(secretion) of monoclonal receptor molecules that bind to the proteinmolecule ligand. This ligand is a portion of the protein to which theimmunogenic polypeptide corresponds in amino acid residue sequence.Thereafter, the hybridoma cells that produce monoclonal receptormolecules that bind to the protein ligand are cultured further toprepare additional quantities of those hybridoma cells, and themonoclonal receptors secreted by those cells that bind to the proteinmolecule ligand. Typically, such culturing is done at limiting dilution,e.g., at an average of about one cell per culture-growing well.

In preferred practice, the hybridoma cells that are prepared are alsoassayed for the production of monoclonal receptor molecules that bind tothe polypeptide immunogen as well as the protein ligand. Thereafter,hybridoma cells that produce monoclonal receptor molecules that bind toboth the immunogenic polypeptide and to the protein ligand are thosecells that are preferably cultured.

Where samples of the protein molecule ligand are limited, it isconvenient to first screen the hybridomas for secretion of monoclonalreceptors that bind to the immunogenic polypeptide. Hybridoma clonesthat exhibit positive binding to that polypeptide are then typicallyfrozen for storage. They are thereafter thawed, and subcloned bylimiting dilution for assurance that truly monoclonal antibodies areproduced, rather than a plurality of monoclonal receptors being producedfrom a plurality of different hybridoma cells. Those limiting dilutionsubcloning cultures are again typically carried out free from feederlayers or macrophages, as such are not necessary.

The hybridoma cells that are ultimately produced may be culturedfollowing usual in vitro tissue culture techniques for such cells as arewell known. More preferably, the hybridoma cells are cultured in animalsusing similarly well known techniques with the monoclonal receptorsbeing obtained from the ascites fluid so generated. The animals used forgeneration of the ascites fluid are typically 129xBALB/c mice bred inthe mouse colony of the Scripps Clinic and Research Foundation, LaJolla, Calif. However, when animals other than mice are used forpreparation of the hybridomas, that animal type is used for theproduction of ascites fluid.

As noted previously, it is preferred that the myeloma cell line be fromthe same species as the receptor. Therefore, fused hybrids such asmouse-mouse hybrids Shulman et al., Nature, 276, 269 (1978)! or rat-rathybrids Galfre et al., Nature, 277, 131 (1979)! are typically utilized.However, some rat-mouse hybrids have also been successfully used informing hybridomas Goding, "Production of Monoclonal Antibodies by CellFusion", in Antibody as a Tool, Marchalonis et al. eds., John Wiley &Sons Ltd., p. 273 (1982)!. Suitable myeloma lines for use in the presentinvention include MPC-11 (ATCC CRL 167), P3X63-Ag8.653 (ATCC CRL 1580),Sp2/0-Agl4 (ATCC CRL 1581), P3X63 Ag8U.1 (ATCC CRL 1597), andY3-Agl.2.3. (deposited at Collection Nationale de Cultures deMicroorganisms, Paris, France, number I-078) and P3X63Ag8 (ATCC TIB 9).Myeloma lines Sp2/0-Agl4 and P3X63-Aq 8.653 are preferred for use in thepresent invention.

Thus, following the method of this invention it is now possible toproduce relatively high yields of monoclonal receptors that bind to orimmunoreact with known, predetermined epitopes of protein molecules suchas oncoproteins. In addition, once the skilled worker has producedhyperimmune serum containing oligoclonal antibodies that exhibit a 50percent binding titer of at least about 1:400 to the immunizingpolypeptide, that worker may follow the before-mentioned steps, take thespleen from the hyperimmunized animal, fuse its antibody-producing cellswith cells of a myeloma line from the same animal type or strain, and besubstantially assured that one or more hybridomas produced from thatfusion secrete monoclonal receptors that bind to the immunizingpolypeptide and to the corresponding protein, such as an oncoprotein.Such results were not heretofore possible.

The above method is useful for preparing hybridomas that secretemonoclonal receptors to substantially any protein molecule ligand.Illustrative of such hybridomas and their monoclonal receptors are thoseraised to immunogenic polypeptides of moderate length whose amino acidresidue sequences correspond to amino acid residue sequences ofoncoproteins encoded by oncogenes. Exemplary oncogenes and usefulimmunogenic polypeptides are shown below followed by the parenthesized,numerical position from the amino-terminus in the oncoprotein sequenceto which the polypeptide corresponds wherein the amino acid residuesequences of those polypeptides are given from left to right and in thedirection of amino-terminus to carboxyterminus, and are represented by aformula selected from the group consisting of formulae shown in Table 1,below:

                  TABLE 1                                                         ______________________________________                                        Poly-                                                                         peptide                                                                             On-                                                                     Number                                                                              cogene.sup.1                                                                           Polypeptide Sequence                                           ______________________________________                                        109   v-sis    DPIPEELYKNLSGHSIRSF (8-26)                                     113   v-sis    RKIEIVRKKPIFKKATV (138-154)                                    114   v-sis    RVTIRTVRVRRPPKGKHRKC (191-210)                                 116   v-sis    TRSHSGGELESLARGKR (50-66)                                      120   v-sis    CKHTHDKTALKETLGA (210-225)                                     110   c-sis    LVSARQGDPIPEELVE (1-16)                                        111   PDGF-1   SIEEAVPAVCKT (1-12)                                            112   PDGF-2   SLGSLTIAEPAMIAECKT (1-18)                                      113   PDGF-2   RKIEIVRKKPIFKKATV (73-89)                                      114   PDGF-2   RVTIRTVRVRRPPKGKHRKC (126-145)                                 121   v-fes.sup.ST                                                                           IGRGNFGEVFSG (519-530)                                         122   v-fes.sup.ST                                                                           IHRDLARRNCLVTEKN (632-647)                                     123   v-fes.sup.ST                                                                           VPVKWTAPEALNYGR (674-688)                                      124   v-fes.sup.ST                                                                           SSGSDVWSFGILLWE (690-704)                                      125   v-fes.sup.ST                                                                           SDVWSFGILLWETFSLGASPYPNLSNQQTR                                                (693-722)                                                      126   v-fes.sup.ST                                                                           SPYPNLSNQQTR (711-722)                                         127   v-fes.sup.ST                                                                           LMEQCWAYEPGQRPSF (744-759)                                     128   v-fes.sup.ST                                                                           CWAYEPGQRPSF (748-759)                                         129   v-fes.sup.ST                                                                           LWETFSLGASPYPNLSNQQTR (702-722)                                131   v-myb    RRKVEQEGYPQESSKAG (2-18)                                       132   v-myb    RHYTDEDPEKEKRIKELEL (94-112)                                   133   v-myb    LGEHHCTPSPPVDHG (160-175).sup.3                                141   v-ras.sup.Ha                                                                           KLVVVGARGVGK (5-16)                                            142   v-ras.sup.Ha                                                                           YREQIKRVKDSDDVPMVLVGNKC (96-118)                               146   v-ras.sup.Ha                                                                           YTLVREIRQHKLRKLNPPDESGPGC (157-181)                            232   v-ras.sup.Ha                                                                           DGETCLLDILDTTGQEEY (47-64)                                     143   v-ras.sup.Ki                                                                           KLVVVGASGVGK (5-16)                                            147   v-ras.sup.Ki                                                                           YTLVREIRQYRLKKISKEEKTPGC (157-180)                             148   v-ras.sup.Ki                                                                           YREQLKRVKDSEDVPMVLVGNKC (96-118)                               144   T24-ras.sup.Hu                                                                         KLVVVGAVGVGK (5-16)                                            145   N-RAS    KLVVVGAGGVGK (5-16)                                            231   N-RAS    DGETCLLDILDTAGQEEY (47-64)                                     237   N-RAS    YTLVREIRQYRMKKLNSSDDGTQGC (157-181)                            233   H-RAS    YKRMKKLNSSDDGTQGC (166-182)                                    234   K-RAS    AGPEAQRLPGLLK (-13 to -1)                                      235   K-RAS    CGDSLAARQGAGRR (-180 to -167)                                  236   ras.sup.K4B                                                                            KHKEKMSKDGKKKKKKSKTKC (165-184)                                149   v-bas    KLVVVGAKGVGK (5-16)                                            150   MYC      APSEDIWKKFELLPTPPLSP (44-63)                                   151   MYC      CDEEENFYQQQQQSEL (25-40)                                       152   MYC      PAPSEDIWKKFEL (43-55)                                          153   MYC      LPTPPLSPSRRSGLC (56-70)                                        154   MYC      CDPDDETFIKNIIIQDC (117-133)                                    155   MYC      CSTSSLYLQDLSAAASEC (171-188)                                   156   MYC      CASQDSSAFSPSSDSLLSSTESSP (208-231)                             157   MYC      CTSPRSSDTEENVKRRT (342-358)                                    158   MYC      SVQAEEQKLISEEDLLRKRR (405-424)                                 159   MYC      LRKRREQLKHKLEQLRNSC (420-438)                                  160   MYC      IIIQDCMWSGFSAA (128-141)                                       182   N-MYC    PPGEDIWKKFELLPTPPLSP (44-63)                                   183   N-MYC    VILQDCMWSGFSAR (110-123)                                       184   N-MYC    SLQAEEHQLLLEKEKLQARQ (432-451)                                 185   N-MYC    LQARQQQLLKKIEHARTC (447-464)                                   192   L-MYC    APSEDIWKKFELVPSPPTSP (44-63)                                   193   L-MYC    IIRRDCMWSGFSAR (110-123)                                       161   v-mos    LPRELSPSVDSR (42-53)                                           162   v-mos    RQASPPHIGGTY (260-271)                                         163   v-mos    TTREVPYSGEPQ (301-312)                                         164   v-mos    IIQSCWEARGLQRPSA (344-359)                                     165   v-mos    LGSGGFGSVYKA (100-111)                                         168   v-mos    TLWQMTTREVPYSGPQYVQYA (296-317).sup.3                          761   v-mos    TLWQMTTREVPYSGEPQYVQY (296-316)                                166   c-mos    IIQSCWEARALQRPGA (344-359)                                     167   MOS      VIQRCWRPSAAQRPSA (316-331)                                     762   MOS      TLWQMTTKQAPYSGERQHILY (268-288)                                171   v-erb B  IMVKCWMIDADSRPKF (366-381)                                     172   v-erb B  LGSGAFGTIYKG (138-149)                                         173   v-erb B  ENDTLVRKYADANAVCQ (23-39)                                      174   v-erb B  VWELMTFGSKPYDGIPASEIS (324-344)                                175   neu      IMVKCWMIDSECRPRF (959-974)                                     178   neu      VWELMTFGAKPYDGIPAREIP (917-937)                                179   neu      LGSGAFGTVYKG (731-742)                                         176   HER-1    RRRHIVRKRTLRRLLQERE (645-663)                                  177   HER-1    VWELMTFGSKPYDGIPASEIS (880-900)                                207   v-src    LLNPENPRGTFLVRESETTKG (162-182)                                208   v-src    TFVALYDYESRTETDLSFKKGERL (85-108)                              202   v-src.sup.PC                                                                           LGQGCFGEVWMG (273-284)                                         205   v-src.sup.PC                                                                           LTELTTKGRVPYPGMVNREVL (452-472)                                201   v-src.sup.PC                                                                           LMCQCWRKDPEERPTF (494-509)                                     203   v-src.sup.SRA                                                                          GSSKSKPKDPSQRRRS (2-17)                                        204   v-src.sup.SRA                                                                          LTELTTKGRVPYPGMGNGEVL (452-472)                                206   SRC      LMCQCWRKEPEERPTF (Note 2)                                      211   v-fgr    AMEQTWRLDPEERPTF (631-646)                                     212   v-fgr    LGTGCFGDVWLG (410-421)                                         213   v-fgr    LTELISKGRVPYPGMNNREVL (589-609)                                214   FGR      LTELITKGRIPYPGMNKREVL                                          215   FGR      LLNPGNPQGAFLIRESETTKG (48-68)                                  221   int-1    DYRRRGPGGPDWHWGGC (154-170)                                    222   int-1    LHNNEAGRTTVFS (200-212)                                        223   int-1    EPEDPAHKPPSPHDL (275-289)                                      224   int-1    RACNSSSPALDGCEL (313-327)                                      240   v-yes    LMKLCWKKDPDERPT (778-792)                                      241   v-yes    LTELVTKGRVPYPGMVNREVL (736-756)                                242   v-yes    VFVALYDYEARTTDDLSFKKGERF (369-393)                             243   v-yes    LLNPGNQRGIFLVRESETTKG (446-466)                                250   v-mil    LVADCLKKVREERPLF (317-332)                                     252   v-mil    VLYELMTGELPYSHINNRDQI (270-290)                                251   v-raf    IGSGSFGTVYRG (355-366).sup.3                                   260   v-raf    LVADCVKKVKEERPLF (285-300)                                     261   v-raf    VLYELMAGELPYAHINNRDQI (237-258)                                253   RAF      IGSGSFGTVYKG (355-366)                                         262   A-RAF    LLTDCLKFQREERPLF (374-389)                                     266   A-RAF    VLYELMTGSLPYSHIGSRDQI (327-347)                                254   PKS      IGTGSFGTVFRG (25-36)                                           255   PKS      VLYELMTGSLPYSHIGCRDQI (207-227)                                256   PKS      LLSDCLKFQREERPLF (254-269)                                     270   v-rel    TLHSCWQQLYSPSPSA (382-397)                                     290   v-fms    LGTGAFGLVVEA (1093-1104).sup.3                                 291   v-fms    LWEIFSLGLNPYPGILVNSKF (1336-1356)                              292   v-fms    FMQACWALEPTRRPTF (1379-1394).sup.3                             293   v-fms    LGTGAFGKVVEA (1078-1089)                                       295   FMS      IMQACWALEPTHRPTF (888-903)                                     296   FMS      LEAGVSLVRVRGRPLMR (134-150)                                    297   FMS      LYVKDPARPWNVLAQE (99-114)                                      298   FMS      VPAELVRIRGEAAQIVC (208-224)                                    310   v-abl    LGGGQYGEVYEG (367-389)                                         311   v-abl    LWEIATYGMSPYPGIDLSQVY (548-568)                                312   v-abl    LMRACWQWNPSDRPSF (590-605)                                     313   c-abl I  KSKKGLSSSSSCYLE (12-26)                                        314   c-abl I  LLSSGINGSFLVRESESSPG (140-159)                                 315   c-abl I  LFVALYDFVASGDNTLSITKGEKL (65-88)                               316   c-abl II DLLSDELHLKLLVLDV (5-20)                                        317   c-abl III                                                                              RWTYTKCRVQRDPALPFM (4-21)                                      318   c-abl IV QQPGKVLGDQRRPSLPALHFIK (3-24)                                  320   BPK C    LGTGSFGRVMLV (48-59)                                           322   BPK C    IYEMAAGYPPFFADQPIQIY (227-246)                                 321   BPK R    DNHGSFGELALM (197-209)                                         323   BPK R    LLRNLLQVDLTKRFGNLK (224-241)                                   340   CDC 28   VGEGTYGVVYKA (14-25)                                           352   v-fps    LWEAFSLGAVPYANLSNQQTR (1110-1130)                              353   c-fps    LMQRCWEYDPRRRPSF (888-903)                                     355   c-fps    NKLAELGSEEPPPALPLQ (484-501)                                   360   v-ros    LGSGAFGEVYEG (254-265)                                         361   v-ros    VWETLTLGQQPYPGLSNIEVL (455-475)                                362   v-ros    LMTRCWAQDPHNRPTF (497-512)                                     366   ROS      IWEILTLGHQPYPAHSNLDVL (362-382)                                367   ROS      LMTQCWAQEPDQRPTF (404-419)                                     371   HIR      LGQGSFGMVYEG (990-1001)                                        372   HIR      LWEITSLAEQPYQGLSNEQVL (1187-1207)                              373   HIR      LMRMCWQFNPNMRPTF (1229-1244)                                   600   TRK      LGEGAFGKVFLA (339-350)                                         601   TRK      LWEIFTYGKQPWYQLSNTEAI (540-560)                                602   TRK      IMRGCWQREPSNATAS (582-597)                                     661   v-kit    LWELFSLGSSPYPGMPVDSKF (637-657)                                662   v-kit    IMKTCWDADPLKRPTF (680-695)                                     701   PKC      LGKGSFGKVMLA (344-355)                                         702   PKC      LYEMLAGQPPFDGEDEDELF (528-547)                                 703   PKC      LMTKHPGKRLGCGPEGE (572-588)                                    711   PKC      LGKGSFGKVMLS (356-367)                                         712   PKC      LYEMLAGQAPFEGEDEDELF (531-550)                                 713   PKC      LITKHPGKRLGCGPEGE (575-591)                                    722   PKC      LYEMLAGQPPFDGEDEEELF (545-564)                                 723   PKC      FLTKHPAKRLGSGPDGE (589-605)                                    771   pim-1    LGSGGFGSVYSG (44-55)                                           772   pim-1    LYDMVCGDIPFEHDEEIIKG (232-251)                                 773   pim-1    LIKWCLSLRPSDRPSF (266-281)                                     841   syn      LGNGQFGEVWMG (277-288)                                         842   syn      LTELVTKGRVPYPGMNNREVL (456-476)                                843   syn      LMIHCWKKDPEERPTF (498-513)                                     844   syn      LFVALYDYEARTEDDLSFHKGEKF (86-109)                              845   syn      LLSFGNPRGTFLIRESETTKG (163-183)                                861   Gs       RLLLLGAGESGK (42-53)                                           862   Gs       RWLRTISVILFLNK (279-293)                                       871   Gi       KLLLLGAGESGK (35-46)                                           872   Gi       KWFTDTSIILFLNK (258-271)                                       882   Go       KFFIDTSIILFLNK (214-227)                                       892   T        RYFATTSIVLFLNK (253-266)                                       894   T'       KFFAATSIVLFLNK (257-270)                                       901   PBK      LGRGVSSVVRRC (25-36)                                           902   PBK      MYTLLAGSPPFWHRKQMLML (219-238)                                 903   PBK      LVSRFLVVQPQKRYTAEE (263-280)                                   911   CGK      LGVGGFGRVELV (365-376)                                         912   CGK      MYELLTGSPPFSGPDPMKTY (547-566)                                 913   CGK      LIKKLCRDNPSERLGNLK (589-606)                                   921   MLCK     LGGGKFGAVCTCT (67-79)                                          922   MLCK     TYMLLSGLSPFLGDDDTETL (248-267)                                 923   MLCK     FVSNLIVKEQGARMSAAQC (292-310)                                  390   c-lsk    LGAGQFGEVWMG (251-262)                                         391   c-lsk    LMMLCWKERPEDRPTF (472-489)                                     392   c-lsk    LTEIVTHGRIPYPGMTNPEVI (430-450)                                393   c-lsk    LVIALHSYEPSHDGDLGFEKGEQL (65-88)                               394   c-lsk    LLAPGNTHGSFLIRESESTAG (141-162)                                400   MET      MLKCWHPKAGMRP (Note 2)                                         401   MET      LWELMTRGAPPYPDVNTFDFI (Note 2)                                 402   MET      VMLKCWHPKAGMRPSF (Note 2)                                      411   FOS      SGFNADYEASSRC (4-17)                                           412   FOS      LSPEEEEKEKRRIRKGTEYETD (132-153)                               413   c-fos    LSPEEEEKRRIRRERNKMMAAKC (132-154)                              414   c-fos    TLQAETDQLEDEKSALQTEI (164-183)                                 415   c-fos    LQTEIANLLKEKEKLEFI (179-196)                                   416   c-fos    RKGSSSNEPSSDSLSSPTLL (359-378)                                 421   TGF-alpha                                                                              VVSAFNDCPDSHTQFC (1-16)                                        423   TGF-alpha                                                                              FHGTCRFLyQEDKPA (17-31)                                        424   TGF-alpha                                                                              HSGYVGVRCEHADL (34-47)                                         431   EGF      NSDSECPLSHDGYC (1-13)                                          432   EGF      CLHDGVCMYIEALDKYAC (15-30)                                     441   bcl-1                                                                   442   bcl-1    RPPQVPAFRRPKSAEPTC                                             443   bcl-1    CITVEGRNRGPG                                                   444   bcl-1    KLMELRIPLSRKSSRGC                                              461   v-erb A  KSFFRRTIQKNLHPTSC (58-75)                                      462   v-erb A  VDFAKNLPMFSELPCEDQ (214-231)                                   463   v-erb A  ELPPRRCRALQILGSILPFV (379-398)                                 470   HGR      KVFFKRAVEGQHNYLCAGR (442-460)                                  471   HGR      NVMWLKPESTSHTLI (728-742)                                      472   HGR      TNQIPKYSNGNIKKLLFHQK (758-777)                                 473   HGR      VKWAKAIPGFRNLHLDDQ (575-592)                                   800   ER       KAFFKRSIQGHNKYMCPA (206-223)                                   801   ER       INWAKRVPGFVDLTLHDQ (358-375)                                   477   cPR      KVFFKRAMEGQHNYLCAGR (Note 2)                                   1000  Beta-TGF ALDTNYCFSSTEKNC                                                ______________________________________                                        .sup.1 Literature citations for the above oncogenes and sequences.            Devare et al., Proc. Natl. Acad. Sci. USA, 79,                                3179-3182 (1982).                                                             Johnsson et al., EMBO J., 3:921-928 (1984).                                   PDGF-1 - Doolittle et al., Science, 221, 275-277 (1983).                      PDGF-2 - Doolittle et al., Science, 221, 275-277 (1983).                      v-fes.sup.ST - Hampe et al. Cell, 30, 775-785 (1982)                          Rushlow et al., Science, 216, 1421-1423 (1982).                               v-ras.sup.Ha - Dhar et al., Science, 217, 934-937 (1982).                     v-ras.sup.Ki - Tsuchida et al., Science, 217, 937-939 (1982).                 T24-ras.sup.Hu - Reddy et al., Nature, 300, 149-152 (1982).                   N-RAS - Taparowski et al., Cell, 34, 581 (1983).                              H-RAS - Capon et al., Nature, 302, 33 (1983).                                 K-RAS -McGrath et al., Nature, 304, 501 (1983).                               ras.sup.K4B - Chardin et al., EMBO J. 5, 2203 (1986).                         Reddy et al., J. Virol, 53, 984 (1985).                                       MYC - Colby et al., Nature, 301, 722-725 (1983).                              N-MYC - Kohl et al., Nature, 319, 73 (1986).                                  L-MYC - Nau et al., Nature, 318, 69 (1985).                                   Van Beveren et al., Nature, 289, 258-262 (1981).                              Van Beveren et al., Cell, 27, 97 (1981).                                      MOS - Watson et al., Proc. Natl. Acad. Sci. USA., 79, 4078                    (1982)                                                                        v-erb-B - Yamamoto et al., Cell, 35, 71 (1983).                               Bargmann et al., Nature, 319, 226 (1986).                                     HER-1 - Ullrich et al., Nature, 309, 418 (1984).                              Takeya et al., J. Virol., 44, 12 (1982).                                      v-src.sup.PC - Schwartz et al., Cell, 32, 853 (1983)                          v-src.sup.SRA - Czernilofsky et al., Nature, 287, 198 (1980).                 SRC - Parker et al., Mol. Cell. Biol., 5, 831 (1985).                         Naharro et al., Science, 223, 63 (1984).                                      1 - Ooyen et al., EMBO J., 4, 2905 (1985)                                     Kitamura et al., Nature, 297, 205 (1982).                                     Sutrave et al., Nature, 309, 85 (1984).                                       Mark et al., Science, 224, 285 (1984).                                        RAF - Bonner et al., Nuc. Acid Res., 14, 1009 (1986).                         A-RAF - Huleihel et al., Mol. Cell. Biol., 6, 2655 (1986).                    PKS - Mark et al., Proc. Natl. Acad. Sci. USA, 83, 6312                       (1986)                                                                        Stephens et al., Proc. Natl. Acad. Sci. USA, 80,                              6229 (1983)                                                                   Hampe et al., Proc. Natl. Acad. Sci. USA, 81, 85                              (1984)                                                                        FMS - Coussens et al., Nature, 320, 277 (1986).                               v-abl I - Reddy et al., Proc. Natl. Acad. Sci. USA, 80,                       3623 (1983)                                                                   c-abl I - Ben-Nerich et al., Cell, 44:577 (1986).                             c-abl II - Ben-Nerich et al., supra.                                          c-abl III - Ben-Nerich et al., supra.                                         c-abl IV - Ben-Nerich et al., supra.                                          BPK C - Shoji et al., Proc. Natl. Acad. Sci. USA, 78, 848                     (1981)                                                                        BPK R - Shoji et al., Biochem., 22, 3702 (1983).                              CDC 28 - Lorincz et al., Nature, 307, 183 (1984).                             Shibuya et al., Cell, 30, 787 (1982).                                         Huang et al., J. Mol. Biol., 181, 175 (1985).                                 Neckameyer et al., S. Virol., 53, 879 (1985).                                 HIR - Ullrich et al., Nature, 313, 756 (1985).                                TRK - Martin - Zanca et al., Nature, 319, 743 (1986).                         Besmer et al., Nature, 320, 415 (1986).                                       PKC - Kropf et al., Cell, 46 491 (1986).                                      pim-1 - Selten et al., Cell, 46, 603 (1986).                                  Semba et al., Proc. Natl. Acad. Sci. USA, 83, 5459                            (1986)                                                                        Gs - Itoh et al., Proc. Natl. Acad. Sci. USA, 83, 3776                        (1986)                                                                        Gi - Itoh et al., supra.                                                      Go - Itoh et al., supra.                                                      T - Itoh et al., supra.                                                       Itoh et al., supra.                                                           PBK - Reimann et al., Biochem., 23, 4185 (1984).                              CGK - Takio et al., Biochem., 23, 4207 (1984).                                MLCK - Takio et al., Biochem., 24, 6028 (1985).                               Marth et al., Cell, 43, 393 (1985).                                           MET - Dean et al., Nature, 318, 385 (1985).                                   FOS - Cochran et al., Science, 226, 1080 (1984).                              van Straaten et al., Proc. Natl. Acad. Sci. USA,                              80, 3138 (1983).                                                              v-erb-A - Weinberger et al., Nature, 318, 670 (1985).                         c-erb-A - Weinberger et al., Nature, 324, 641 (1986).                         HGR - Hollenberg et al., Nature, 318, 635 (1985)                              ER - Greene et al., Science, 231, 1150 (1986).                                CPR - Connelly et al., Science, 233, 767 (1986).                              Beta-TGF Derynck et al., Nature, 316, 701 (1985).                             .sup.2 Numerical position of the polypeptide was not available                because the sequence reported was incomplete.                                 .sup.3 These polypeptides contain a deleted, added or substituted             amino acid residue as compared to the reported sequences.                 

The homologous polypeptides encoded by the above four ras genes may beconveniently written as one amino acid residue sequence, from left toright and in the direction from amino-terminus to carboxy-terminus,represented by the formula

    KVVVGAR(S,V,G)GVGK

wherein the amino acid residues in parentheses are each an alternativeto the immediately preceding amino acid residue, "R", in the formula.

Still further useful polypeptides for inducing the production ofmonoclonal receptors of this invention are the polypeptides whoseoncogene, position in the oncoprotein sequence and polypeptide aminoacid residue sequences are shown in FIGS. 20, 21, and 22. Thosepolypeptides correspond to sequence-conserved regions in the well knownfamily of protein kinase oncoproteins, some of whose oncogenes have beenpreviously noted herein.

II. MONOCLONAL RECEPTORS

While the present invention contemplates a large number of monoclonalreceptors, only a relatively few of those contemplated receptors, in theform of intact monoclonal antibodies (Mabs), will be discussed in detailherein as illustrative of the group. The before-discussed test for theimmunogenicity and antigenicity of a polypeptide will be discussedthereafter for polypeptides corresponding to additional monoclonalreceptors that bind (immunoreact) to different oncoproteins.

A. Exemplary Receptors

Using the procedures discussed herein, exemplary monoclonal receptorswere raised to oncogene-related polypeptides.

Hybridomas secreting monoclonal receptors of the invention have beendeposited at the American Type Culture Collection (ATCC) in Rockville,Md. pursuant to the Budapest Treaty. A list of those deposits includingtheir ATCC accession number (ATCC No.), laboratory reference number(Ref. No.), date of receipt at the ATCC (ATCC Receipt), and the numberof the immunizing polypeptide cross-referenced to the polypeptides ofTable 1 (Polypep. No.) is provided in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        ATCC Deposits                                                                 ATTC No. Ref. No.   ATCC RECEIPT                                                                              POLYPEP No.                                   ______________________________________                                        B 8593   P44E11.sup.1                                                                             08/02/84    125                                           HB 8594  P43D09     08/02/84    125                                           HB 8595  S22C06     08/02/84    125                                           HB 8596  S10F03     08/02/84    125                                           HB 8679  1/24-24E05 12/12/84    142                                           HB 8800  18-9B10    05/09/85    112                                           HB 8888  133-1E10   08/15/85    133                                           HB 8894  173-1C11   08/27/85    173                                           HB 8895  202-11AB   08/27/85    202                                           HB 8896  173-8D2    08/27/85    173                                           HB 8897  133-6C10   08/27/85    133                                           HB 8898  203-7D10   08/27/85    203                                           HB 8899  203-6F5    08/27/85    203                                           HB 8900  202-9D10   08/27/85    202                                           HB 8924  132-7C9    08/29/85    132                                           HB 8925  114-50D4   08/29/85    114                                           HB 8926  114-50G2   08/29/85    114                                           HB 8927  132-1C8    08/29/85    132                                           HB 8948  121-1F9    12/03/85    121                                           HB 8949  121-3H5    12/03/85    121                                           HB 8950  121-4F8    12/03/85    121                                           HB 8951  121-5E5    12/23/85    121                                           HB 8952  121-9G10   12/03/85    121                                           HB 8953  121-9E5    12/03/87    121                                           HB 8954  121-15B10  12/03/85    121                                           HB 8955  121-19B10  12/03/85    121                                           HB 8956  121-8D8    12/04/85    121                                           HB 8965  127-24C7   12/11/85    127                                           HB 8966  127-24E11  12/11/85    127                                           HB 8967  127-38G2   12/11/85    127                                           HB 8968  127-50D4   12/11/85    127                                           HB 8969  127-50D12  12/11/85    127                                           HB 8970  127-53F8   12/11/85    127                                           HB 8971  127-60F3   12/11/85    127                                           --       127-42C11  --          127                                           HB 8976  155-11C7   12/17/85    155                                           HB 8996  152-6D11   01/28/86    152                                           HB 8997  146-3E4    01/28/86    146                                           HB 8998  146-17A3   01/28/86    146                                           HB 8999  146-8D11   01/28/86    146                                           HB 9000  155-9F6    01/28/86    155                                           HB 9001  155-8G1    01/28/86    155                                           HB 9002  310-5F5    01/28/86    310                                           HB 9003  131-94H4   01/28/86    131                                           HB 9004  172-12G7   01/28/86    172                                           HB 9005  172-12A4   01/28/86    172                                           HB 9040  164-3F3    03/19/86    164                                           HB 9052  222-35C8   03/27/86    222                                           HB 9053  310-29F7   03/27/86    310                                           HB 9077  133-10F6   04/17/86    133                                           HB 9097  171-19B10  05/08/86    171                                           HB 9098  171-10E5   05/08/86    171                                           HB 9117  171-11B9   05/29/86    171                                           HB 9133  2904E10    06/26/86    290                                           HB 9144  240-13D10  07/10/86    240                                           HB 9208  312-13E08  09/24/86    312                                           HB 9227  361-31C05  10/15/86    316                                           HB 9260  250-9G06   11/06/86    250                                           HB 9278  147-67C6   11/20/86    147                                           HB 9279  165-34E4   11/20/86    165                                           HB 9280  360-27E06  11/20/86    360                                           ______________________________________                                         .sup.1 Hybridoma P44E11 was prepared using the myeloma cell line P3X63Ag      8.653. All other hybridomas were prepared using the myeloma cell line         SP20, as discussed in the Materials and Methods section.                 

Five exemplary hybridomas secreting monoclonal receptors were raised tothe v-fes related, 30-mer immunogenic, synthetic polypeptide shown below(polypeptide number 125 also referred to as polypeptide a), and eachalso binds to the carboxy-terminal 12-mer polypeptide shown below(polypeptide 126 also referred to as polypeptide b), as well as bindingto the oncoprotein denominated p85 (85K daltons) encoded by the v-fesgene of ST-FeSV. Those hybridomas were given the reference numbersS10F03, S22C06, P43D09, P42C10 and P44E11 These hybridomas have thedesignations ATCC HB 8596, ATCC HB 8595, ATCC HB 8594, ATCC HB 8593,ATCC HB 8679, respectively. The amino acid residue sequences ofsynthetic polypeptides (a) and (b), from left to right and in thedirection from amino-terminus to carboxy-terminus, are represented bythe formulae

    polypeptide a SDVWSFGILLWETFSLGASP-YPNLSNQQTR;

    polypeptide b SPYPNLSNQQTR.

The seven hybridomas deposited at the ATCC of Table 2 that were raisedto the v-res-related polypeptide number 127 and are shown in Table 1 areamong the nineteen hybridomas raised to that polypeptide. The monoclonalreceptors secreted by those seven hybridomas also bind to the p85oncoprotein.

The monoclonal receptors of this invention secreted by hybridomasdesignated S22C06 and S10F03 are particularly preferred monoclonalreceptors. Both preferred monoclonal receptors are IgG1 monoclonalreceptors, having kappa light chains, that immunoreact with theimmunizing polypeptide and with the fes-related oncoprotein having anamino acid residue sequence corresponding to the sequence of theimmunizing polypeptide.

A hybridoma was raised using the ras 23-mer immunogenic, syntheticpolypeptide number 142 (ras) shown below:

    YREQIKRVKDSDDVPMVLVGNKC.

The monoclonal antibody secreted by that hybridoma binds to theimmunogenic polypeptide and also binds to the 55K dalton protein encodedby the ras gene of the Harvey sequence. The monoclonal antibodyrecognizes a 23K dalton protein in all ras-producing cell lines testedas well as a higher molecular weight protein.

The hybridomas designated S10F03, S22C06, P43D09, P44E11 and 1/24/E05secrete kappa-light chained, IgG1 monoclonal receptors. These hybridomashave the designations ATCC HB 8596, ATCC HB 8595, ATCC HB 8594, ATCC HB8593, ATCC HB 8679, respectively.

The last-named five hybridomas were prepared from three separate cellfusions. The efficiency of producing hybridomas whose Mabs recognize theimmunogenic polypeptide as well as the corresponding oncoproteinmolecule ligand for the first preparation was 100 percent; i.e., twoMabs (from S10F03 and S22C06) were produced that recognize thepolypeptide, and those two Mabs also recognize the oncoprotein. For thesecond and third preparations, the efficiency, calculated similarly wasabout 20 percent.

Another hybridoma was raised using the erb-B related, 16-mer immunogenicsynthetic polypeptide number 171 shown below. The amino acid residuesequence of the synthetic polypeptide, from left to right and in thedirection from amino-terminus to carboxy-terminus is represented by theformula:

    IMVKCWMIDADSRPKF.

The monoclonal antibody secreted by this hybridoma also binds topolypeptides related to oncoproteins encoded by fes, fms, abl, src andfgr oncogenes. The erb-B related hybridomas have ATCC designations ATCCHB 9097, ATCC HB 9098 and ATCC HB 9117.

FIG. 1 illustrates the immunological detection of the p85 oncoproteinligand by the monoclonal receptors secreted by hybridomas S10F03 (ATCCHB 8596) and S22C06 (ATCC HB 8595), using an external standard for thep85 oncoprotein ligand and an influenza hemagglutinin-recognizing Mab asa negative control. FIG. 2 illustrates similar results again using Mabsfrom hybridoma S10F03 as well as Mabs from hybridomas P43D09 (ATCC HB8594), and P44E11 (ATCC HB 8593), and also hybridoma P42C10. Amonoclonal antibody against the Rauscher virus protein denominated gp70Niman and Elder in Monoclonal Antibodies and T Cell Products, above! wasused as a negative control.

FIG. 3 further illustrates the specificity of the monoclonal receptorsof this invention. There, CCL64 mink cells (lanes B and C) or MSTF cellsinfected with FeLV-B and FeSV (lanes A and B) were radioactively labeledwith ³² p. Extracts from the labeled cells were then incubated witheither a goat antiserum against the p15 protein encoded by the gagportion of the v-fes gene and expressed as the protein precursordenominated pr65 (lanes A and B) or with tissue culture supernatant fromhybridoma S10F03 (lanes C and D) (ATCC HB 8596).

As can be seen, the Mab of this invention from hybridoma S10F03 boundonly to the p85 oncoprotein ligand (lane C), while the goat anti-p15serum bound to both the pr65 and p85 fusion oncoproteins from theinfected cells (lane A). No proteins were bound from the uninfectedcells (lanes B and D). These results and, by analogy, discussion of theassay concerning FIG. 13, confirm that the Mabs of this invention bindonly to the oncoprotein ligand (p85) a portion of whose amino acidresidue sequence corresponds to the sequence of the immunogenicpolypeptide used to prepare the hybridoma secreting each Mab.

In similar results, not shown, Mabs from the above five hybridomas alsobound to the 108K dalton oncoprotein ligand expressed in cellstransformed by GA-FeSV. The oncoprotein ligand encoded by the GA-FeSVstrain is substantially identical in amino acid residue sequence to theoncoprotein ligand encoded by the ST-FeSV strain in the region of theimmunogenically useful polypeptide. See, Hampe et al., Cell, 30, 777-785(1982).

None of the above five Mabs bound to the oncoprotein encoded by thev-fps gene of the Fujinami strain of avian sarcoma virus. The predictedv-fps oncoprotein, whose sequence is reported by Shibuya et al., Cell,30, 787 (1982), also contains extensive homologies to the predictedv-fes oncoprotein and differs in the region corresponding to the above12-mer (polypeptide b) only by the substitution of the first and fourthresidues from the amino-terminus of that 12-mer polypeptide; i.e., theamino-terminal serine (S) of the v-fes-related polypeptide andoncoprotein is replaced by a valine (V) in the v-fps-relatedoncoprotein, and the second proline (P) residue from the amino-terminusis replaced by an alanine (A) residue.

The non-binding of the above Mabs to the v-fps-related oncoproteinprovides a basis for distinguishing among expressed oncoproteins intransformed cells, and for assaying for the presence of thev-fes-related oncoprotein ligand in the presence of the v-fps-relatedoncoprotein. That distinction in binding can also be useful in purifyinga mixture of both proteins by affinity chromatography utilizing an Mabof this invention as a portion of an affinity sorbant, as is discussedhereinafter.

The above non-binding of the monoclonal antibodies of this invention tothe v-fps-related oncoprotein also highlights the improvement inspecificity of the monoclonal receptors over previously obtainedoligoclonal receptors. Thus, Sen et al., Proc. Natl. Acad. Sci. USA, 80,1246-1250 (1983), used polypeptide (b) above conjugated to KLH toprepare rabbit oligoclonal antibodies. Those oligoclonal antibodiesbound to oncoproteins expressed in cells transformed by ST-FeSV, GA-FeSVand FSV (Fuginami sarcoma virus) that contain the v-fes^(ST), v-fes^(GA)and v-fps oncogenes, respectively. It can therefore be seen that thespecificity obtained from the monoclonal receptors of this invention isgreatly improved over that obtained with oligoclonal receptors even whenboth are raised to the same immunogenic polypeptide.

In a similar manner are prepared hybridomas that secrete monoclonalreceptors that bind to oncoprotein molecule ligands, e.g., PDGF, toimmunogenic polypeptides encoded by the retroviral oncogenes denominatedfes, myb, fos, sis, ras, myc and mos, as well as to immunogenicpolypeptides whose sequences correspond to sequences of oncoproteinsencoded by oncogenes denominated fps, src, yes, fgr, bas, int-1, fms,erb-A, erb-B, mil, raf (mil/raf), abl and ros, as well as growth factorsPDGF1, PDGF-2, EGF, TGF-alpha and also to oncoproteins expressed incells transformed by retroviruses containing those genes. Specificmonoclonal receptors of this invention bind to an immunogenicpolypeptide encoded by the above oncogenes.

Some of those oncogenes are named below in Table 3 and are illustratedadjacent to polypeptide numbers correlated to the oncogenes, sequencesand polypeptide numbers of Table 1 to which the preferred monoclonalreceptors of this invention bind. Data relating to the binding of atleast one monoclonal receptor (Mab) or oligoclonal antiserum (serum)raised to each polypeptide in a Western blot analysis are also providedin Table 3 adjacent to the polypeptide number.

                  TABLE 3                                                         ______________________________________                                        Receptor Binding To Oncoproteins.sup.1                                                  Polypep. Mab Binding   Serum Binding,                               Oncogene  No.      To Oncoprotein.sup.3                                                                        To Oncoprotein.sup.4                         ______________________________________                                        sis       110      +             NT                                                     111      +             +                                                      112      +             +                                                      113      NT            +                                                      114      +             +                                            fes       121      +             +                                                      122      NT            NT                                                     123      NT            +                                                      124      NT            NT                                                     125      +             +                                                      126      NT            +                                                      127      +             +                                            myb       131      +             +                                                      132      +             +                                                      133      +             NT                                           ras       141      NT            +                                                      142      +             +                                                      143      NT            +                                                      144      NT            NT                                                     145      NT            +                                                      146      +             NT                                                     147      +             NT                                           bas       149      +             NT                                           myc       151      NT            +                                                      152      +             +                                                      153      NT            +                                                      154      NT            NT                                                     155      +             +                                                      156      NT            NT                                                     157      NT            +                                            mos        16      1NT           +                                                      162      NT            +                                                      163      NT            +                                                      164      +             NT                                                     165      +             NT                                           erb-B     171      +             +                                                      172      +             NT                                                     173      +             +                                            src       201      +             NT                                                     202      +             +                                                      203      +             NT                                           fgr       211      NT            NT                                                     213      +             NT                                           int-.sup.1                                                                              221      NT            NT                                                     222      +             NT                                           yes       240      +             NT                                                     241      +             NT                                           mil       250      +             NT                                           raf       251      +             NT                                           fms       290      +             NT                                                     292      +             NT                                           abl       310      +             NT                                                     311      +             NT                                                     312      +             NT                                           ros       360      +             NT                                                     361      +             NT                                           fos       411      +             NT                                                     413      +             NT                                                     416      +             NT                                           TGF-alpha 421      +             NT                                           erb-A     461      +             NT                                                     462      +             NT                                           ______________________________________                                         .sup.1 Binding of receptor molecules to oncoproteins in Western blot          analyses. Plus signs (+) indicate that binding was shown. NT = not tested     .sup.2 Polypep. No. = polypeptide number from Table 1.                        .sup.3 Binding of a monoclonal receptor molecules to an oncoprotein.          .sup.4 Binding of oligoclonal antipolypeptide serum to an oncoprotein.   

The polypeptides useful for inducing the production of oligoclonalreceptors, and ultimately for production of monoclonal receptors, arepreferably linked to a carrier molecule, as discussed herein whereinpolypeptides linked to KLH have been utilized throughout as illustrativepolypeptide-carrier conjugates. For polypeptides that contain fewer thanabout 35 amino acid residues, it is preferable to use a carrier for thepurpose of inducing the production of oligoclonal and monoclonalreceptors. Polypeptides containing about 35 to about 40 amino acidresidues may be used alone, without linkage to a carrier, to inducereceptor production, although it is still preferable to utilize acarrier for producing those receptors. Thus, the receptors may beinduced by or raised to a polypeptide alone, or linked to a carrier.

B. Immunization Binding Studies

As noted several times, the polypeptides utilized in raising oligoclonalantibodies and hybridomas that secrete monoclonal antibodies arethemselves immunogenic and antigenic, and those properties providecriteria for identifying useful polypeptides for hybridoma preparation.The discussion below relates to studies with oligoclonal antibody(receptor) containing antisera induced by or raised to polypeptides usedin the preparation of hybridomas that secrete monoclonal receptors(antibodies) to oncoproteins encoded by the ras, sis erb-B and myboncogenes. As will be described, the sis-related polypeptide inducesproduction of oligoclonal receptors that bind not only to thepolypeptide, but also to a corresponding oncoprotein, humanplatelet-derived growth factor (PDGF). The oligoclonal antibodies soprepared exhibited the before described 50 percent binding titer to theimmunizing polypeptide, thereby indicating that monoclonal antibodies(receptors) of this invention may also be prepared by fusion of theantibody-producing splenocytes with cells of suitable myeloma line.

PDGF isolated from platelets consists of two chains that areapproximately sixty percent homologous at the amino-terminal end. One ofthose chains (PDGF-2) is virtually identical to a portion of the simiansarcoma virus (v-sis) gene product (p28^(sis)). Sequencing of the humanc-sis and v-sis terminate at the same position and the PDGF-2 moleculeoriginates from a larger precursor which has extensive homology withp28^(sis). The homology between p28^(sis) and PDGF-2 begins at aminoacid residue 67 of p28^(sis) and the amino-terminus of PDGF-2, and hasrecently been extended to the predicted carboxy-terminus of p28^(sis)via the isolation and sequencing of a human c-sis clone. Josephs et al.,Science, 223, 487-491 (1984).

p28^(sis) is rapidly cleaved to generate p20^(sis) which presumably hasthe same amino terminus as PDGF-2. Within the region coding forp20^(sis) and PDGF-2 there are eight amino acid changes that can beplaced into three regions. The two changes near the amino-terminus areconservative, five changes are clustered near the center of themolecule, and one change is located in the carboxyl-terminal portion.

Two exemplary polypeptides were prepared. The first, denominatedpolypeptide number 113 also referred to as polypeptide (c), correspondsin amino acid residue sequence to residues 139 through 155 of thepredicted sequence of the simian sarcoma virus transforming proteindenominated p28^(sis). Devare et al., Proc. Natl. Acad. Sci. USA, 80,731-735 (1983). The sequence of polypeptide (c) also corresponds to thesequence of positions 73 through 89 from the amino-terminus of theprotein chain denominated PDGF-2 of human platelet-derived growthfactor, as noted before. The second, denominated polypeptide number 131also referred to as polypeptide (d), corresponds in amino acid residuesequence to residues 2 through 18 of the predicted sequence of thetransforming protein of the avian myeloblastosis virus (v-myb)oncoprotein. Rushlow et al., Science, 216, 1421-1423 (1982). The aminoacid residue sequence of polypeptides (c) and (d) are shown below, fromleft to right and in the direction from amino-terminus tocarboxy-terminus: ##STR2##

Each of the polypeptides was synthesized and bound to KLH using a Cysresidue of their carboxy-termini (not shown in the above formulas), andeach resulting conjugate was then used to immunize mice as discussedgenerally in the Materials and Methods section. As can be seen from anexamination of FIG. 4, sera raised to polypeptide (c) containedoligoclonal receptors that bind to polypeptide as well as to KLH, andsera raised to polypeptide (d) contained oligoclonal receptors that bindto polypeptide (d) and to KLH. Neither serum contained receptors thatcross-react and bind to the polypeptide not used to raise them.

Extracts from outdated human platelets were used to obtain partiallypurified samples of PDGF. As already noted, PDGF is an oncoproteinhaving an apparent molecular weight of about 30K daltons that can bereductively cleaved into two high molecular weight polypeptides ofsimilar apparent molecular weights, and designated PDGF-1 and -2.

FIG. 5 shows the results of Western blot analysis of PDGF using theoligoclonal receptor-containing antisera raised to polypeptides (c) and(d), as is discussed in more detail in the description of that figure;the antiserum raised to polypeptide (d) being used as a negativecontrol. As can be seen from an examination of FIG. 5; the oligoclonalreceptor-containing serum raised to the sis-related polypeptide,polypeptide (c), bound to three proteinaceous moieties (lane 2). One ofthose moieties has an apparent molecular weight of about 30K daltons andtwo of about 16-18K daltons each. Lane 4 also illustrates binding byoligoclonal receptors contained in the anti-sis related polypeptideserum. As expected, only non-specific binding was shown by oligoclonalreceptors raised to the myb-related polypeptide, polypeptide (d), (lanesand 5).

Presuming that the amino acid residue sequence of PDGF-1 and -2 arecollinear with the sequence of p28^(sis), the amino acid residuesequence of the polypeptide (c) corresponds to positions 67 through 83,and 73 through 89 of PDGF-1 and -2, respectively. The amino acid residuesequence of residues 73 through 80 of PDGF 2 has been determinedDoolittle et al., Science, 221, 275-277 (1983)! and all of the thoseresidues are identical to the first (amino-terminal) eight residues ofpolypeptide (c). In addition, a polypeptide from PDGF and correspondingto residues 147 through 155 of the p28^(sis) oncoprotein has beensequenced Waterfield, Nature, 304, 35-39 (1983)!, and of the nineresidues so far identified, all are identical to the correspondingresidues of polypeptide (c). Thus, sixteen of the seventeen residues ofpolypeptide (c) are identical to and in the same sequence as residues inboth PDGF, derived from humans, and p28^(sis) derived from a line ofretrovirus-transformed cells.

The above results thus illustrate the immunogenicity and antigenicity oftwo additional polypeptides useful for immunizations leading to thepreparation of hybridomas that secrete monoclonal receptors of thisinvention. Those results also show that the oligoclonal receptors raisedto polypeptide (c) also bind to an oncoprotein; i.e., PDGF, PDGF-1 andPDGF-2.

Additional synthetic polypeptides representing various regions of bothPDGF sequences were made. The amino-termini of PDGF-1 and PDGF-2, aswell as the central and carboxy-terminal portion of PDGF-2 weresynthesized, conjugated to the immunogenic carrier keyhole limpethemocyanin (KLH), and injected into mice to induce production ofoligoclonal receptor-containing antisera that exhibited thebefore-described 50 percent binding titer.

The polypeptide representing the unique region of PDGF-2 contains thefirst 18 amino acid residues of this sequence and will be calledPDGF-2(1-18) (polypeptide number 112), wherein the parenthesizednumerals indicate the amino acid residues of the corresponding moleculenumbered from amino-terminus. The unique region of PDGF-1 is representedby a polypeptide PDGF-1(1-12) also referred to as polypeptide number111, that contains the first 12 amino acids of that sequence. Six ofthose 12 amino acids are shared with PDGF-2 but only three areconsecutive, as noted before. The third polypeptide, PDGF-2(73-89) isalso referred to herein as polypeptide (c) and polypeptide number 113.It represents the predicted amino acid residues 139-155 of p28^(sis) andcontains an additional cysteine at its carboxy-terminus for couplingpurposes. This polypeptide when coupled to KLH induced production ofantibodies that recognize the reduced subunits of purified PDGF,proteins of MW 31,000, 30,000, 21,000 and 18,000-14,000 in a plateletextract, and a 56K dalton protein in SSV-infected marmoset cells. Thefourth polypeptide, PDGF-2(126-145), was also predicted by the v sissequence (residues 191-210 of p28^(sis) also referred to as polypeptide114). Amino acid sequences of these polypeptides have been illustratedhereinbefore.

To analyze the specificity of the oligoclonal receptor-containingantisera generated against these synthetic polypeptide conjugates, PDGFwas probed with these antisera. Purified PDGF was reduced andelectrophoresed into a polyacrylamide gel, and then onto nitrocellulose(FIG. 6, lanes A-F) using a Western blot procedure. In lanes A and B,two antisera directed against PDGF-1(1-12) immunoreacted with a proteinof approximately 18,000 daltons. Sequence analysis of purified PDGFindicates the majority of the PDGF-1 chain migrates at this positionAntonaides, et al., Science, 220, 963-965 (1983)!. The weakness of thereactivity with these antisera suggests the amino-terminal end of PDGF-1may not be readily accessible for antibody binding.

In contrast, antiserum against the amino-terminus of PDGF-2 (1-18) (laneC) readily detected a protein migrating at about 18,000 and 14,000daltons, consistent with sequence analysis of PDGF-2 (Antonaides et al.,supra.).

The antisera induced by PDGF-2(73-89) produced the same activities(lanes D, E) as seen in lane C. In contrast, antisera againstPDGF-2(126-145) did not have detectable activity against purified PDGF.

Since the sequence of the PDGF-2(126-145) polypeptide differs fromc-PDGF at position 145 (Josephs, et al., supra), it is possible thatthis amino acid residue change is contained within the epitopic site.This is unlikely because the polypeptide is 20 amino acid residues longand the change is only on the carboxy-terminal position that is used tocouple the polypeptide to the KLH carrier protein. The lack of activityis thus not due to generation of oncopolypeptide specific antibodiesbecause this antiserum reacts with cell-derived PDGF-like molecules. The14,000 to 18,000 dalton size of the detected PDGF in purifiedpreparations suggest that most of this material is missing thecarboxy-terminal end of the predicted sequence of p28^(sis), which wouldremove all or part of the PDGF antigenic site recognized by thisantiserum.

In order to determine if PDGF-like proteins might also be synthesized inother transformed cell lines, extracts were made and immunoreacted withvarious oligoclonal receptor containing antisera against PDGF-relatedpolypeptides. In FIG. 7, the SSV-transformed NIH 3T3 cells were probedwith an oligoclonal receptor-containing antiserum induced byPDGF-1(1-12) (lanes A-C, F-H and K-M) and by PDGF-2(73-89) (lanes D, E,I, J, N and O). Of the two sera against PDGF-2(73-89) (FIG. 6, lanes Dand E), the serum used in FIG. 6, lane D produced a somewhat weakeractivity with purified PDGF. However, as seen in lane D of FIG. 7, astrong reactivity with a protein of approximately 70,000 daltons wasobserved that was blocked by preincubation with the immunizingpolypeptide, PDGF-2(73-89) (lane E), but was not blocked bypreincubation of the antiserum with PDGF-1(1-12).

Thus, the specific reactivity with these oncoproteins by both antiserademonstrates that this is not a fortuitous cross-reactivity with a smallregion of PDGF, but that this molecule contains sequences homologous toat least the amino-terminus of PDGF-1 and the central region of PDGF-2.The amounts of p28^(sis) and p20^(sis) were below the level of detectionwith this anti-PDGF-2(73-89) serum. Similar results were obtained withadditional antisera, although overexposure did occasionally show a20,000 dalton band was specifically detected (data not shown).

Analysis of extracts of two other unrelated transformed cells with theseantisera gave similar results. The TRDl cell line is a spontaneouslytransformed Balb/3T3 cell line Bowen-Pope et al., Proc. Natl. Acad. Sci.USA, 81, 2396-2400 (1984)!. This line also expresses a 70,000 daltonprotein as well as a more immunologically related protein ofapproximately 100,000 daltons (FIG. 7, lanes G-I). A third cell line,MSTF, and a mink lung line (CCL64) productively infected with FeLV-B andthe Snyder-Theilen strain of FeSV, also expresses the same size proteinFIG. 7, lanes K-O.

In addition to the 70,000 dalton oncoprotein, an oligoclonalreceptor-containing antiserum against PDGF-1(1-12) detected proteins ofapproximately 53,000 daltons (data not shown). These proteins are notserum contaminants because they are detected in extracts of cells thathave been grown for one month in the absence of serum and are found inserum free media conditioned by the TRDl cell lines. All cell linesstudied contain these two PDGF-like proteins. (See also discussion ofFIG. 11 in "Brief Description of Figures").

The expression of PDGF-like molecules in a broad spectrum of cells,including cells that are not oncogenically transformed (normal diploidrat smooth muscle and human lung ribroblasts), indicates that otherprocesses are involved in transformation. Although all of the cell linescontained 70,000 and 53,000 dalton proteins detected with oligoclonalreceptor-containing antisera induced by PDGF-1(1-12), the cells werequite heterogeneous with regard to size and intensity of other proteinsdetected with antisera directed against determinants predicted by thesequence of the PDGF-2 region (data not shown). The nature of thesedifferences is presently unknown.

In a similar manner, each of the four immunogenic polypeptides,denominated (e-h) below, may be used to induce oligoclonal receptorsthat bind to those immunogenic polypeptides used to induce theirproduction as well as to each of two oncoproteins encoded by the rasoncogene. The sequences of those four ras-related polypeptides, in thedirection from left to right and from amino-terminus tocarboxy-terminus, are represented by the formulas:

    ______________________________________                                        polypeptide e KLVVVGARGVGK (polypeptide 141);                                 polypeptide f KLVVVGASGVGK (polypeptide 143);                                 polypeptide q KLVVVGAVGVGK (polypeptide 144);                                 polypeptide h KLVVVGAGGVGK (polypeptide 145);                                 ______________________________________                                    

    or

by the combined formula:

    ______________________________________                                                 polypeptide (e-h)                                                             KLVVVGAR(S,V,G)GVGK;                                                 ______________________________________                                    

wherein the amino acid residues in parentheses are each an alternativeto the immediately preceding amino acid residue in the formula. Theoligoclonal receptors so prepared have a 50 percent binding titerdilution of more than 1:400 after two immunizations, as describedbefore, in about a one month period. Additionally, each ras-relatedoligoclonal receptor induced by polypeptides (e), (f) and (h) have beenshown to bind to an oncoprotein present in lysed cell extracts from (a)human T24 bladder carcinoma cells and also (b) Harvey murine sarcomavirus-infected mouse 3T3 cells (data not shown).

As is seen in FIG. 12, each of the two immunogenic polypeptidesdenominated below (k and l) may be used to induce oligoclonal receptorsthat bind to those immunogenic polypeptides used to induce theirproduction as well as to each of two oncoproteins encoded by thevfes^(ST) oncogene. The sequence of the two v-fes-related polypeptides,in the direction from left to right and from amino-terminus tocarboxy-terminus are represented by the formulae:

    ______________________________________                                        polypeptide k LEMQCWAYEPGQRPSF                                                (polypeptide 127);                                                            polypeptide 1 IGRGNFGEVFSG                                                    (polypeptide 121).                                                            ______________________________________                                    

The oligoclonal receptors induced by polypeptides (k) and (l) have beenshown to bind to an oncoprotein present in supernatant from cells ofhuman T24 bladder carcinoma and a spontaneously transformed mouse 3T3cell line (Lanes A and C).

Monoclonal receptors secreted by hybridomas ATCC HB 8952, HB 8954 and HB8955 raised to polypeptide 121 have been shown to immunoreact with oneor more proteins obtained from tumors of the breast, rectum, stomach andendometrium. Reactivity of a monoclonal receptor raised to polypeptide127 (hybridoma 127-42C11) with proteins in urine samples of pregnantmothers is discussed hereinafter.

As shown in FIG. 13, a protein related to the ras oncogene was detectedby a monoclonal antibody (from hybridoma ATCC HB 8679) raised to arassynthetic peptide corresponding to positions 96-118 of v-ras^(Ha)(polypeptide 142). The protein is detected in lane A and blocked bypreincubation with the immunizing peptide (lane B). Thus, thepreincubation with the immunizing polypeptide blocked the stronglyreactive oncoprotein.

The use of monoclonal receptors of this invention such as those raisedto the sis-(PDGF) related polypeptide (c), or to the fes-relatedpolypeptides (a), (b) (k) or (l), or to the ras-related polypeptides(e-h) or to the other oncoprotein-related polypeptides disclosed hereinin the affinity sorbants described below provides a convenient and lessarduous means for preparing naturally occurring proteinaceous materialsthat are otherwise difficult to obtain in purified form such as PDGF.Thus, rather than having to go through the long procedure to obtainpurified PDGF, discussed hereinafter, one may, for example, merely lysethe cells, centrifuge, pour the supernatant through an affinity sorbantcolumn containing bound anti-polypeptide (c) receptor, and elute thepurified protein after dissociating the formed, reversible ligandcomplex. While some additional proteinaceous material may benon-specifically bound to the affinity sorbant column, the isolation ofpurified proteins that are otherwise difficult to obtain in such form isgreatly enhanced using such sorbants.

The antisera to the conserved sequences described above react withproteins in a wide variety of transformed cell lines. The antiserareadily detected oncogene-related proteins that were five-to-fifty-foldmore concentrated in the urine of cancer patients and pregnant womenthan in normal controls. Unique patterns of expression were detected invarious malignancies and during different gestational stages ofpregnancy.

Anti-peptide antibodies are particularly suited for detecting proteinsimmunologically related to sequenced oncogenes Wong et al., Proc. Natl.Acad. Sci. USA, 78, 7412-7416 (1981)!. Since they are sequence specific,anti-peptide antibodies can be directed toward highly conserved regionsof proteins to maximize the probability of identifying related moleculeswhich may have similar functions. Because immune recognition of proteinsby anti-peptide antibodies need not be highly dependent upon antigenconformation, one can identify proteins that are not detected byanti-protein antibodies, the bulk of which are directed againstdeterminants unique to the folded protein. Finally, the binding ofanti-peptide antibodies is relatively insensitive to alteration orfragmentation of the target antigen such as might occur in bodily fluidsor secretions.

In Tables 1 and 3, the synthetic peptides used to generate theantibodies are enumerated and listed together with related sequences ofother oncogenes. An exemplary ras polypeptide 142 is the v-ras^(Ha)sequence located at 37-59 amino acids downstream from the threonineresidue auto-phosphorylated by p21 encoded by v-ras^(Ha) or v-ras^(Ki).The sequence is identical in H-RAS and N-RAS, and differs from K-RAS byone conservative amino acid change. Capon et al. Nature 304, 507 (1983).The sequence of PDGF-2 used to generate the sis monoclonal antibodies islocated at the amino-terminus of the chain (polypeptide 112) and ishomologous to the first 12 amino-acids of the other chain (PDGF-1) ofplatelet-derived growth factor. The fes peptide (polypeptide 127)constitutes residues 744-759 of the 85,000 dalton fusion protein ofv-fes-st (positions 927-942 of v-fes-GA) and is 79-94 amino acidsdownstream from the major tyrosine phosphorylation site. The peptidesused for this study were selected because they represent highlyconserved regions of the respective oncogene families.

The antisera to these conserved sequences react with proteins in a widevariety of transformed cell lines. The reactivity of the three antiserawith proteins of a mink lung line transformed by feline sarcoma virusare shown in FIG. 14. Antibodies against the sis-peptide detect a 20,000dalton protein in SSV-transformed NRK cells as well as a sis-relatedprotein of approximately 56,000 daltons (p56^(sis)) in the mink lungline (lane 1). Antibodies against the ras peptide detect a major proteinof approximately 21,000 daltons (p21^(ras)) and a minor protein ofapproximately 30,000 daltons in the cell extract (lane 2). The antiserumagainst the fes protein detects the 85,000 dalton gag-fes fusion protein(pp85-^(gag-ges)) as well as a 40,000 dalton protein (p40^(fes), lane3).

In FIG. 15, the reactivity of these antisera with urinary proteins froma variety of patients is demonstrated. The sis antisera detect proteinsof 56,000, 31,000 and 25,000 daltons in urine concentrates (Panel A).

The antibody binding to all three proteins is blocked by priorincubation with the sis peptide (Panel B) but not by incubation with theras peptide (Panel A). The concentrations of the detected proteins arefive to fifty fold higher than normal individuals (see below). Allurines studied contained the three sis-related proteins except for thesample from the patient with lymphoma which is missing the 56,000 daltonprotein (lane 4).

The somewhat faster mobilities of p56^(sis) (Panel A, lanes 1 and 2) inthe urine from the donors with multiple myeloma and gastric cancer isdue to excess albumin in these samples, whereas the distortion of thelower molecular weight proteins in lane 1 are due to excessive amountsof antibody light chain.

In Panel C the various ras-related proteins detected in urine samplesare displayed. Proteins are approximately 100,000 and 55,000 daltons aredetected (Panel C, lane 2-4). Again, the specificity of the antiserumwas demonstrated by blocking the activity by preincubation with the raspeptide (Panel D) but not by preincubation with the sis peptide (PanelC).

The 55,000 dalton ras-related protein is different from the 56,000dalton sis-related protein (see below) and displays different reactivitypatterns in each sample. The protein is not detectable in Panel C, lane1 (gastric cancer) while four bands of almost equal intensity are seenin lane 2 (38 weeks pregnant).

A strongly reactive doublet is visualized in lane 3 when urine from apatient (donor) with breast cancer was probed. A minor band atapproximately 35,000 daltons is associated with high concentrations ofthe 55,000 dalton protein. In lane 4, a single 55,000 dalton band wasdetected.

Proteins of approximately 21,000 daltons were detected in all 4 lanes ofPanel C. These smaller proteins were present at similar concentrationsalthough the mobility of the protein in Panel C, lane 1 is slightlyslower. This altered mobility may be significant because of the effectof changes at amino acid residue position 12 on the electrophoreticmobility of ras encoded proteins. The binding detected at 25,000 daltonsis difficult to interpret due to comigration with antibody light chain.

In Panel E, the 35,000 and 40,000 dalton fes-related proteins are shown.The binding was blocked by preincubation with the immunizing fes peptide(panel E, lane 1) but not incubation with the ras peptide or peptidesrepresenting the homologous sequences in erb B or abl proteins (Panel E,lanes 2-4).

In summary, the 3 antisera described above specifically detect 8different proteins in urine, 3 sis-related proteins (p56^(sis),p31^(sis), and p25^(sis)), 3 ras-related proteins (p100^(ras),p55^(ras), and p21^(ras)) and 2 fes-related proteins (p40^(fes),p35^(fes)).

In FIG. 23, the frequencies of detection of oncogene-related proteins inurine samples of the 51 control (normal; free from diagnosed neoplasticdisease) or 189 urine samples from patients (donors) with a variety ofmalignancies are listed. Similar frequencies in 260 urine samples frompregnant women are shown in FIG. 24. The amount of oncogene-relatedproteins in the urine was estimated using immunoblots, and was placedinto 1 of four categories: undetectable, detectable, 5-15-fold elevated,and greater than 15-fold elevated.

The types of malignancies in which more than 10 samples were tested arelisted individually. The remaining types are listed as a composite.

p21^(ras) was detected in approximately 70% of all tumor samples.However, similar frequencies were found in apparently normalindividuals. In contrast to the elevated levels of the ras- andfes-related proteins found in urine of breast cancer patients, bladderand prostate cancer patients frequently secrete elevated levels of the56,00 dalton sis-related protein. This protein was detected in theabsence of the ras- and fes-related proteins described above (FIG. 15,lanes 1, 2, Panels A-C). In addition to the 56,000 dalton sis-relatedprotein, these patients frequently had elevated levels of the 31,000and/or 25,000 sis-related proteins. In further contrast, urine from apatient with a benign prostate nodule did not contain elevated levels ofthese oncogene-related proteins (FIG. 18, lane 3, Panels A-C).

High levels of the smaller proteins were also found frequently in urinefrom patients with lung and cervical cancer as well as non-Hodgkinslymphomas (see FIG. 23). In these latter patients, the elevated 31,000and/or 25,000 sis-related proteins were found in the absence of the56,000 dalton protein (FIG. 5, lane 4, Panel A-B).

Thus, in the urine samples from cancer patients three unusual patternshave been observed. A subset of the breast cancer patients have elevatedlevels of p55^(ras) in conjunction with p40^(fes) and/or p35^(fes).Patients with bladder and prostate cancer excrete increased amounts ofall three sis-related proteins in the absence of p55^(ras), p40^(fes),and p35^(fes). Finally, a subset of lung cancer and lymphoma patientsexcreted elevated levels of only the lower molecular weight sizes thesis-related proteins. As can be seen from FIGS. 15-18 as well as FIG.23, patterns of expression correlate with disease states better thanexcretion of high levels of a single oncogene-related protein. Inapparently normal individuals, elevated levels of these proteins arerarely detected.

The proteins described herein are immunologically related to oncogeneproteins based upon the highly specific reactivity of the variousanti-peptide antisera. However, of the eight proteins described, onlytwo (p21^(ras) and p31^(sis)) represent oncogene-encoded whole proteins.

The p21^(ras) protein has GTP binding activity. Thus, p21^(ras) isintimately involved with cell division and therefore it is notsurprising that the protein is readily detected in most urine samples.

Similarly, elevated levels of transcripts specific for H-ras or K-rashave been detected in a wide variety of malignancies as is shown herein.Furthermore, antisera to ras-related products have also detectedelevated expression in tumor tissues. Here, the most striking elevationof this protein was found in the urine of malignancies.

p31^(sis) protein, which is one of the chains of the platelet-derivedgrowth factor (PDGF), was also detected. Although PDGF-1 chain is only18,000 daltons when isolated from platelets, comparison of the humanc-sis sequence with v-sis indicates the 18,000 dalton protein originatesfrom a larger precursor protein. Indeed, analysis of a partiallypurified platelet extract reveals a protein of approximately 31,000daltons. Since PDGF has potent mitogenic activity and is released fromplatelets at the site of tissue injury, one of the physiologicalfunctions of PDGF is thought to be wound healing. In addition, PDGF-likematerial is secreted from a number of transformed cell lines andsecretion appears to be developmentally regulated in smooth musclecells. Thus, p31^(sis) like p21^(ras) may be physiologically important,and it is not surprising that it is present in the urine in normal andabnormal states.

In addition to the oncogene encoded proteins of expected molecular size,additional proteins were detected in this study. It is not likely thattheir presence is due to spurious cross-reactivities since they areuniquely present in certain cancers as well as during pregnancy.Further, the reaction of the antibodies with these proteins wasinhibited specifically with the appropriate synthetic immunogens. Sincethe peptides used as immunogens represent conserved sequences amongoncogene families, these additional proteins may represent members ofthese gene families. The expression of these genes may come undercoordinate control during neoplasia or pregnancy. Regardless of theorigin of these proteins, the fact that they are uniquely expressedduring neoplasia and pregnancy makes them important markers.

III. DIAGNOSTIC SYSTEMS AND METHODS

A diagnostic system, preferably in kit form, comprises yet anotherembodiment of this invention. This system is useful for assaying for thepresence of an oncoprotein ligand by the formation of an immunereaction. This system includes at least one package that containsbiologically active monoclonal receptor molecules of this invention.Thus, the receptor binds to (a) a polypeptide containing about 7 toabout 40, and preferably about 10 to about 30, amino acid residues in anamino acid residue sequence that corresponds to a portion of the aminoacid residue sequence of an oncoprotein ligand encoded by a gene of aretrovirus, and (b) the oncoprotein ligand encoded by a retroviral gene.

When a predetermined amount of monoclonal receptor molecules is admixedwith a predetermine amount of an aqueous composition containing anoncoprotein ligand, an immunological reaction occurs that forms acomplex between the receptor and the ligand (antibody and antigen).Exemplary aqueous compositions containing an oncoprotein include,without limitation, cell lysates, serum, plasma, urine and amnioticfluid.

In addition, it is particularly valuable to utilize a screening withantisera to more than one oncogene-related translation product. Thus,assay methods set forth herein can be performed on a group of body fluidsample antiquots taken from a single donor to yield accurate informationregarding a neoplastic state, gestational stage or the like.

Admixture between receptor and ligand occurs in an aqueous composition.However, either the receptor or ligand can be substantially dry andwater-free prior to that admixture. Thus, a solution of the receptor inhybridoma supernatant, ascites fluid or buffer can be admixed with anaqueous cell extract to admix the reagents from two aqueouscompositions; the receptor can be coated on the walls of a microliterplate and then admixed with a cell extract or serum containing theligand; or the ligand can be coated on microliter plate walls, on anitrocellulose sheet after transfer from an acrylamide gel or the like,or can be present in a tissue section, and hybridoma supernatant,ascites fluid or a buffer solution containing the receptor admixedtherewith.

The use of exemplary diagnostic systems and methods of this invention isillustrated in the descriptions of the Figures. There, oncoproteinligands coated onto nitrocellulose and then admixed with a receptor ofthis invention are discussed in relation to FIGS. 1, 2, 5-8, and 11-14,while a cell extract incubated with hybridoma supernatant to form animmunological complex is discussed regarding FIG. 3. Oncoproteins fromurine samples are discussed in FIG. 9, 10 and 15-19.

Receptors are utilized along with an "indicating group" or a "label".The indicating group or label is utilized in conjunction with thereceptor as a means for determining whether an immune reaction has takenplace and an immunological complex has formed, and in some instances fordetermining the extent of such a reaction.

The indicating group may be a single atom as in the case of radioactiveelements such as iodine 125 or 131, hydrogen 3, sulfur 35, carbon 14, orNMR-active elements such as fluorine 19 or nitrogen 15. The indicatinggroup may also be a molecule such as a fluorescent dye like fluorescein,rhodamine B, or an enzyme, like horseradish peroxidase (HRP) or glucoseoxidase,or the like.

The indicating group may be bonded to the receptor as where an antibodyis labeled with ¹²⁵ I. The indicating group may also constitute all or aportion of a separate molecule or atom that reacts with the receptormolecule such as HRP-linked receptor was raised in a mouse, or where aradioactive element such as ¹²⁵ I is bonded to protein A obtained fromStaphylococcus aureus.

Where the principal indicating group is an enzyme such as HRP or glucoseoxidase, additional reagents are required to visualize the fact that animmune reaction has occurred and the receptor-ligand complex has formed.Such additional reagents for HRP include hydrogen peroxide and anoxidation dye precursor such as diaminobenzidine. Additional reagentsuseful with glucose oxidase include ABTS dye, glucose and HRP.

The terms "indicating group" or "label" are used herein to includesingle atoms and molecules that are linked to the receptor or usedseparately, and whether those atoms or molecules are used alone or inconjunction with additional reagents. Such indicating groups or labelsare themselves well-known in immunochemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelreceptors, methods and/or systems.

An indicating group or label is preferably supplied along with thereceptor and may be packaged therewith or packaged separately.Additional reagents such as hydrogen peroxide and diaminobenzidine mayalso be included in the system when an indicating group such as HRP isutilized. Such materials are readily available in commerce, as are manyindicating groups, and need not be supplied along with the diagnosticsystem. In addition, some reagents such as hydrogen peroxide decomposeon standing, or are otherwise short-lived like some radioactiveelements, and are better supplied by the end-user.

The diagnostic system may also include a solid matrix that may be 96well microliter plates sold under the designation Immulon II (Dynatech,Alexandria, Va.). The microliter strip or plate is made of a clearplastic material, preferably polyvinyl chloride or polystyrene.Alternative solid matrices for use in the diagnostic system and methodof this invention include polystyrene beads, about 1 micron to about 5millimeters in diameter, available from Abbott Laboratories, NorthChicago, Ill.; polystyrene tubes, sticks or paddles of any convenientsize; and polystyrene latex whose polystyrene particles are of a size ofabout 1 micron and can be centrifugally separated from the latex.

The solid matrix may also be made of a variety of materials such ascross-linked dextran, e.g. Sephadex G-25, -50, -100, -200, and the likeavailable from Pharmacia Fine Chemicals of Piscataway, N.J., agarose andcross-linked agarose, e.g., Sepharose-6B, CL-6B, 4B CL46 and the likealso available from Pharmacia Fine Chemicals.

The diagnostic system may further include a standard against which tocompare the assay results and various buffers in dry or liquid form for,inter alia, washing microliter plate walls, diluting the sample,diluting the labeled reagent, or the like.

An assay method for the presence of an oncoprotein ligand in a bodysample from a warm-blooded animal constitutes another aspect of thepresent invention. In accordance with the general assay method, amonoclonal receptor of this invention is admixed in an aqueouscomposition that contains the sample to be assayed for the presence ofan oncoprotein ligand. Preferably, the monoclonal receptor and bodysample are utilized in predetermined amounts. The admixture so preparedis maintained for a period of time sufficient for an immunoreaction tooccur between the receptor and ligand and an immunocomplex (reactionproduct or immunoreactant) to form. The presence of an immuno-complex isthen determined, and its presence indicates the presence of theoncoprotein ligand in the assayed sample. The presence of animmunocomplex is determined using the beforedescribed labels or by othermeans well known in immunochemistry for determining the presence of theantibody-antigen complexes.

Specific assay methods are also contemplated. Each of those specificmethods utilizes the above three steps, but the specifics of those assaymethods differ slightly from one another.

Solid phase assays wherein the sample to be assayed is affixed to asolid phase matrix such as a microtiterplate test well or anitrocellulose sheet to form a solid support are particularly preferred.In such instances, admixture of the sample to be assayed and themonoclonal receptor forms a solid-liquid phase admixture. The solid andliquid phases are separated after the before-described maintenanceperiod, and the presence of a liquid-receptor complex is determined bythe presence of receptor bound to the solid support. The relative amountof bound receptor can be determined in many assays, thereby alsoproviding a determination of the amount of oncoprotein ligand that waspresent in the sample assayed.

A receptor molecule of this invention can also be affixed to the solidmatrix to form a solid support. In that instance, the sample to beassayed is admixed to form a solid-liquid phase admixture, the admixtureis maintained as described before, and the presence of an immunocomplexand oncoprotein in the assayed sample are determined by admixture of apredetermined amount of a labeled ligand such as a polypeptide oroncoprotein that is bound by the affixed receptor molecule. Thus, thepresence of a complex formed between the receptor and oncoprotein of thesample provides an amount of labeled ligand binding that is less than aknown, control amount that is exhibited when the sample is free ofoncoprotein being assayed. The relative amount of oncoprotein in thesample can be determined by using an excess of the receptor andmeasuring the lessened binding of the labeled ligand.

A polypeptide or oncoprotein ligand bound by a receptor molecule of thisinvention can also be affixed to a solid matrix to form the solidsupport antigen. A known, excess amount of receptor molecules of thisinvention is admixed with the sample to be assayed to form a liquidadmixture. The liquid admixture so formed is maintained for a period oftime sufficient to form an immunocomplex reaction product, and isthereafter admixed with the solid support to form a solid-liquid phaseadmixture. That admixture is maintained for a period sufficient for theexcess, unreacted receptor molecules present to immunoreact and form acomplex with the solid phase support antigen. The amount of that complexthat is formed is determined, after separation of the solid and liquidphases, using a previously described technique. This method can providea determination as to the presence of oncoprotein in the sample, andalso as to its relative amount, where predetermined amounts of receptorand solid phase ligand are used.

IV. DIFFERENTIAL ASSAY

Liquid body samples can be screened with antisera to more than oneoncogene-encoded protein. The screening can be systematicallyaccomplished in accordance with the assay methods of this invention. Thescreening of samples with more than one antiserum provides a pattern ofoncoproteins present in the sample assayed.

In breast cancer patients, p55^(ras) and p40^(fes) are found to beelevated (FIGS. 16 and 17) in contrast to the p56^(sis) found in bladderand prostate cancer patients (FIG. 18). Also, bladder and prostatecancer patients often demonstrated elevated levels of the 31K dalton or25K dalton sis-related proteins. In contrast, a donor with a benignprostate nodule did not demonstrate these elevated levels of protein.

High levels of the smaller proteins were also found in patients withlung and cervical cancer as well as non-Hodgkins lymphomas (See FIG.23). In these patients, the elevated 31K dalton and/or 25K daltonsis-related proteins were found in the absence of the 56K dalton protein(See FIG. 15, lane 4, Panels A-B).

Thus, in the urine samples from cancer patients three unusual patternshave been observed. A subset of the breast cancer patients have elevatedlevels of p55^(ras) in conjunction with p40^(fes) and/or p35^(fes). Incontrast, patients with bladder and prostate cancer excrete increasedamounts of all three sis-related proteins in the absence of p55^(ras),p40^(fes), and p35^(fes). Finally, a subset of lung cancer and lymphomapatients excrete elevated levels of only the lower molecular weightsizes of the sis-related proteins. As can be seen from the Figures,patterns of expression correlate with diseased states better thanexcretion of high levels of a single oncogene-related protein. Inapparently normal individuals, elevated levels of these proteins arerarely detected.

The finding of oncogene-related proteins in urine was unexpected and hasnot been previously reported by others. This finding provides a basisfor still another method aspect of the present invention.

In accordance with this method, a sample of urine or a urine concentrateis admixed in an aqueous composition, as described before, with areceptor that immunoreacts with an oncoprotein. The admixture ismaintained for a period of time sufficient for an immunocomplex to form,and the presence of an immunocomplex is determined as described beforein relation to the general assay method and the before-describedspecific methods.

In this method, any receptor known to immunoreact with an oncoproteincan be used. Thus, the receptor molecules can be of polyclonal,oligoclonal or monoclonal origin, and can have been raised to a whole orfusion oncoprotein, or a polypeptide as described herein.

Blotting techniques such as those of the Western blots of the Figuresand so-called "slot blots" wherein the sample is affixed to anitrocellulose matrix as a solid support and where the receptormolecules in a liquid aqueous composition are admixed on thenitrocellulose sheet are preferred techniques for analysis. However,other techniques such as solid phase ELISA and radioimmunoassay (RIA)that utilize microliter plate wells as solid matrices, and dip stickmethods are also useful.

V. IN UTERO FETAL SEX DETERMINATION

Five site-directed monoclonal antibody probes and one oligoclonal serumprobe were used to detect oncoprotein ligands related to beta-TGF, EGF,int-1, fes, ras, and myb, in urine from newborn infants and pregnantwomen. A subset of the beta-TGF-related oncoprotein ligands was foundexclusively in newborn female urine samples. A subset of these samplescontain the fes- and ras-related proteins which were elevated in urinefrom breast cancer patients, discussed before. Urine samples from maleand female newborn infants or pregnant women contained additionaloncogene-related proteins. Two proteins (p55^(1ras) and p40^(fas) wereelevated in urine samples from expectant mothers carrying 16-18 weekfemale fetuses. The hybridomas and the synthetic polypeptides used togenerate the antibody probes are listed in Table 4. The samples werescreened using Western blot techniques as are discussed hereinafter.

                  TABLE 4                                                         ______________________________________                                        Site-Directed Antibodies.sup.1/                                               Oncogene/                Polypeptide                                          Growth Factor  Hybridoma Number                                               ______________________________________                                        Beta-TGF       (oligoclonal)                                                                           1000                                                 EGF            432-25G07 432                                                  int-1          222-35C08                                                                     222-33A05 222                                                  fes/FES        127-42C11 127                                                  src            203-07D10 203                                                  H-RAS/N-RAS    142-24E05 142                                                  c-MYC/L-MYC    152-06D11 152                                                  v-myb          133-10F06 .sup. 133.sup.2/                                     ______________________________________                                         .sup.1/ Oncogenes growth factors, hybridoma designations and polypeptide      numbers are as listed in Tables 1 and 2.                                      .sup.2/ This polypeptide is lacking one of the three adjacent histidines      found in the Avian sequence.                                             

Each urine sample was reduced, boiled, and applied to a polyacrylamidegel, as discussed in the Materials and Methods Section. After transferto nitrocellulose, separate samples were probed with each of the sixantisera. The results for twenty-five individuals are listed in Table 5,in which relative density values were estimated optically.

                                      TABLE 5                                     __________________________________________________________________________    PROTEIN LEVELS IN NEWBORN URINE.sup.1                                         FES         V-myb    H/N-RAS                                                                              int-1       Beta-TGF                              PATIENT                                                                             40                                                                              38                                                                              35                                                                              150                                                                              55                                                                              53                                                                              50                                                                              100                                                                              55                                                                              21                                                                              70                                                                              50                                                                              43                                                                              38                                                                              30                                                                              25                                                                              67                                                                              42                                                                              24                                                                              18                                                                              12                            __________________________________________________________________________    KIM   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0 1 0 0                             IBA   0 0 1 0  1 0 0 1  0 1 0 1 0 0 0 2 0 0      5                                                                      0      0                            VAL   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      1                                                                      0      0                            CIN   0 0 0 0  2 0 0 1  0 1 0 0 1 1 0 1 0 0      4                                                                      0      1                            MUN   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      3                                                                      0      0                            ADE   0 0 0 0  1 0 0 0  0 0 0 0 0 0 0 0 0 0      4                                                                      0      0                            BRO   2 1 2 0  1 1 1 1  3 1 0 0 1 1 1 2 2 1      5                                                                      0      2                            IME   2 2 2 1  0 0 1 1  3 2 0 1 0 0 0 2 2 0      5                                                                      3      0                            STR   2 2 2 0  0 0 0 2  4 2 0 1 0 0 0 1 3 0      4                                                                      0      1                            WOL   3 0 3 0  0 0 0 2  5 3 0 0 0 0 0 0 3 0      5                                                                      2      0                            SER   2 1 2 2  3 0 2 0  2 2 0 3 1 1 2 3 1 0      3                                                                      0      1                            CAR   3 2 3 0  1 1 0 0  5 2 0 0 0 1 2 2 2 0      4                                                                      0      2                            TOTAL 6 5 7 2  6 2 3 6  6 8 0 4 4 4 3 7 6 1      12                                                                     2      5                            POS..sup.2                                                                    (FEMALE)                                                                      HOL   0 0 0 0  1 1 0 0  0 1 1 1 0 0 0 1 0 1      0                                                                      0      0                            SAN   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      2                            CEA   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      1                            CAR   0 0 0 0  1 0 0 0  0 1 0 1 0 0 0 0 0 0      0                                                                      0      1                            PEA   0 0 0 0  1 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      2                            SUM   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      1                            MAR   0 0 0 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      1                            BAT   0 0 0 0  2 2 0 0  0 1 0 0 1 1 0 1 0 0      0                                                                      0      2                            MAR   0 0 0 0  2 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      3                            SIM   0 0 0 2  2 0 0 0  0 0 0 2 0 0 2 0 0 0      0                                                                      0      3                            BOO   0 0 9 0  0 0 0 0  0 0 0 0 0 0 0 0 0 0      0                                                                      0      1                            PLE   0 0 0 0  1 1 0 0  0 0 0 0 0 0 0 2 0 0      0                                                                      0      2                            DES   0 0 0 0  4 0 0 0  0 1 0 0 0 0 1 1 0 0      0                                                                      0      2                            TOTAL 0 0 0 1  8 3 0 0  0 4 1 3 1 1 2 4 0 1      0                                                                      0      12                           POS..sup.2                                                                    (MALE)                                                                        __________________________________________________________________________     .sup.1 Oncogenes growth factors, hybridoma designations and polypeptide       numbers are as listed in Tables 1 and 2.                                      .sup.2 This polypeptide is lacking one of the three adjacent histidines       found in the Avian sequence.                                             

                                      TABLE 6                                     __________________________________________________________________________    PROTEINS IN MATERNAL URINE.sup.1                                                       FES      V-myb                                                                             H/N-RAS  int-1                                                                             EGF                                                                              Beta-TGF                                PATIENT  150                                                                              45                                                                              40                                                                              35                                                                              60                                                                              45                                                                              100                                                                              55                                                                              23                                                                              21                                                                              52                                                                              25                                                                              150                                                                              24                                                                              12                                    __________________________________________________________________________    ROB      0  0 0 0 4 2 0  0 0 2 0 0 3  4 2                                     IME      0  3 0 0 3 0 2  0 0 2 4 3 2  0 0                                     GOU      0  2 0 0 2 1 0  0 0 2 2 2 3  3 3                                     ABB      1  1 1 1 0 3 1  4 0 1 0 3 0  0 1                                     GOO      3  3 0 0 1 3 2  0 0 3 3 3 5  3 2                                     VIC      2  2 0 0 0 0 0  0 0 1 3 3 0  NT                                                                              NT.sup.3                              RAS      2  3 3 3 0 2 0  4 0 1 2 2 5  4 1                                     PER      1  1 0 0 4 3 2  2 0 4 0 0 2  4 2                                     MEZ      1  1 1 2 1 2 0  3 1 2 1 3 4  4 1                                     MST      0  1 2 2 0 0 0  4 2 1 0 0 3  3 1                                     BLA      1  1 1 1 2 1 2  2 0 2 3 3 1  NT                                                                              NT                                    TER      2  2 2 2 3 1 2  3 0 2 1 3 3  NT                                                                              NT                                    NIM      2  2 1 1 3 3 1  3 0 2 4 3 4  NT                                                                              NT                                    STR      2  1 0 0 0 3 1  0 0 1 3 3 0  NT                                                                              NT                                    TOTAL    10 13                                                                              7 7 9 11                                                                              8  8 2 14                                                                              8 8 11 7                                       POS..sup.2                                                                    (FEMALE FETUS)                                                                DUQ      0  0 0 0 2 2 0  0 0 2 2 2 3                                          BOO      0  0 0 0 0 1 0  0 0 1 0 2 0                                          MAC      3  2 0 0 1 2 1  0 0 2 0 3 4                                          ESP      0  1 0 3 0 3 1  0 0 3 3 2 2                                          LOR      1  1 0 1 0 0 0  0 0 0 0 2 2                                          BEL      3  2 0 2 0 2 1  0 0 2 4 0                                            HAR      1  2 0 0 1 3 0  0 0 3 2 3                                            WAT      0  2 0 0 1 2 1  0 0 2 2 2                                            TOTAL    4  6 0 3 4 7 4  0 0 7 5 7                                            POS..sup.2                                                                    (MALE FETUS)                                                                  __________________________________________________________________________     .sup.1,2 See notes 1 and 2 of Table 5                                         .sup.3 NT  Not tested.                                                   

Although several oncogene- or growth factor-related proteins werereadily detected, none of the samples contained detectable levels ofproteins related to EGF. However, all of the samples contained proteinsreactive with the oligoclonal antiserum directed against beta-TGF.Moreover, only female samples contained detectable levels of p24/p23beta-TGF (in the presence or absence of pl2 beta-TGF), whereas urinesamples from male newborns had only pl2 beta-TGF.

Sex-associated expression of oncogene-related proteins is also shown inTable 5. A subset of the female samples contained readily detectablelevels of p35^(fes), p38^(fes), p40^(fes), and p100^(ras), p55^(ras),p35^(fes), p40^(fes), and p55^(ras) had been previously detected atelevated levels in urine from a subset of breast cancer patients as wellas pregnant women, as discussed before.

The patterns of oncogene-related proteins in fetal urine displayedconsiderable heterogeneity, although some of the proteins were uniformlydetected in a sex-associated manner. To further characterize theseproteins, sequential collections of concentrated maternal urine wereprobed.

Sequentially collected (between 16-20 weeks) from twenty-two pregnantmothers were also screened by Western blot analysis using thebefore-mentioned six antibody probes. The results of that screening areshown in Table 6. As can be seen, most of the proteins detected innewborn urine are also found in maternal urine. The concentration ofmost of the proteins remained constant during weeks 16-20, althoughunique, patterns were found for each individual. The differences inpatterns were most easily identified by comparing proteins that wereuniformly detected in the 16-20 week time period.

In addition to the proteins that were relatively constant in samplesfrom the same individual, the concentrations of other proteins changeddramatically. For example, p24/p23 beta-TGF was detected in urine frommost of the individuals. In contrast, p40^(fes), and p55^(ras) weredetected only in urine from mothers carrying female fetuses. However,weekly urine collections from all patients with female fetuses did notcontain detectable levels of these proteins.

During the one-month collection period, most of the proteins listed weredetected in approximately half of the patients irrespective of the sexof the fetus. In contrast to these uniformly detected proteins,p40^(fes) and p55^(ras) were exclusively detected in patients carryingfemale fetuses. Urine from eight of the patients contained detectablelevels of p55^(ras) and urine from seven of those eight patients alsocontained detectable levels of p40^(fes). The lack of detection of thesex-associated proteins in urine from all maternal and newborn patientscould be due to short periods of expression. Daily collections ofmaternal urine during the 16-20 week period indicated the proteins weredetected for less than one week.

The short periods of detection may be due to hormonal regulation ofexpression. Initial assays of diabetic pregnant patients receivinginsulin revealed the presence of these proteins over extended periods oftime (at least six weeks). Moreover, the detectability of the proteinswas not dependent upon the sex of the fetus. Similarly, collections fromnormal patients with younger or older male fetuses exhibited thepresence of p55^(ras) Thus, the sex-associated proteins may be maternalproteins induced by external factors or the temporal expression of fetalgenes.

The above results with urine samples from pregnant (expectant) mothersprovide a means for predicting the sex of the fetus being carried. Asnoted before, expectant mothers carrying male fetuses did not expressthe p40^(fes) or the p55^(ras) proteins, whereas expectant motherscarrying female fetuses expressed one or both of those proteins duringthe 16-20-week period of their pregnancies. Some of those expectantmothers carrying female fetuses did not express either of those proteinsin that time period.

Since there were no false positives for expectant mothers carrying malefetuses, the finding of an express p40^(fes) and/or p55^(ras)oncoprotein ligand in the urine of an expectant mother in the first16-20-week pregnancy period provides a positive, noninvasive assay forascertaining the presence of a female fetus in utero. The absence of anexpressed p40^(fes) and/or p55^(ras) oncoprotein ligand in a urinesample of an expectant mother during the 16-20-week period is about50-60 percent (7 of 14 and 8 of 14 samples, respectively) predictivethat the expectant mother is carrying a male fetus.

In accordance with this method, a sample of urine from a pregnant motherin about the first 16 through 20 weeks of her pregnancy is provided, andis preferably reduced as with 2 mercaptoethanol, boiled, and, mostpreferably concentrated. That sample is admixed with receptor moleculesthat immunoreact with a polypeptide that has a formula, from left toright and in the direction from amino-terminus to carboxy-terminus,selected from the group consisting of

    (i) LMEQCWAYEPGQRPSF (polypeptide 12 of Table 1);

and

    (ii) YREQIKRVKDSDDVPMYLVGNKC (polypeptide 142 of Table 1).

The resulting admixture is maintained for a time period sufficient forthe receptor molecules to immunoreact with an oncoprotein ligand in theurine. The presence of an immunoreaction is determined between thosereceptor molecules with an oncoprotein ligand that has a relativemolecular mass in a 5-17 percent polyacrylamide gel of (i) about 40Kdaltons for the receptor molecules that immunoreact with polypeptide(i), above, or (ii) about 55K daltons for the receptor molecules thatimmunoreact with polypeptide (ii), above. The presence of a female fetusin utero.

In preferred practice, the receptor molecules utilized are monoclonal.Most preferably, the monoclonal receptor molecules are secreted byhybridomas having reference numbers 127-42Cll and 142-24E05 (HB 8679),respectively.

VI. AFFINITY SORBANTS

Affinity sorbants in which the monoclonal receptor molecules of thisinvention constitute the active, binding portions constitute yet anotherembodiment of this invention.

In this embodiment, the monoclonal receptor molecules of this inventionare linked to a solid support that is chemically inert to theoncoprotein ligands to be purified by those sorbants. The phrase"chemically inert" is used herein to mean that a chemical reactionbetween the solid support and the oncoprotein ligands does not occur.However, physical interactions between the solid support and theoncoprotein ligands such as non-specific binding can and do occurbetween them, although such interactions are preferably minimized.

The solid support may be made of a variety of materials such ascross-linked dextran, e.g., Sephadex G-25, -50, -100, -200 and the likeavailable from Pharmacia Fine Chemicals of Piscataway, N.J., agarose andcross-linked agarose, e.g., Sepharose 6B, CL6B, 4B, CL4B and the likealso available from Pharmacia Fine Chemicals or Bio-Gel A-0.5M, A-1.5M,A-50M and the like available from Bio-Rad Laboratories, Richmond Calif.,or polyacrylamide beads, e.g., Bio-Gel P-2, P-30, P-100, P-300 and thelike also available from Bio-Rad Laboratories. Polyacrylamide beads havethe lowest tendency for non-specific binding among the above supports,but also typically have a low porosity that limits their bindingcapacity. The agarose and cross-linked agarose materials are preferredherein and will be used illustratively as a solid support.

The agarose support is typically activated for linking cyanogen bromide.The activated support is then washed and linked to the receptormolecules without drying of the activated support. The support-linkedreceptor is then washed and is ready for use. Unreacted reactive groupson the support can be reacted with an amine such as ethanolamine orTris, if desired, although those reactive groups decay quickly.

The affinity sorbant may be used in its loose state, as in a beaker orflask, or it may be confined in a column. Prior to use, it is preferablethat the affinity sorbant be washed in the buffer or other aqueousmedium utilized for oncoprotein purification to eliminatenon-specifically bound proteins or those receptors that were unstablylinked to the support.

An aqueous composition containing an oncoprotein ligand having an aminoacid residue sequence corresponding to the amino acid residue sequenceof the polypeptide to which the linked receptor of the affinity sorbantbinds such as serum or a cell extract is provided, and then and thenadmixed with the affinity sorbant. That admixture forms a reversible,linked receptor-ligand complex between the linked receptor and theoncoprotein ligand.

The ligand receptor-ligand complex is then separated from the remainderof the uncomplexed aqueous composition to thereby obtain the oncoproteinin purified form linked to the affinity sorbant. When the admixturetakes place in a beaker or flask, this separation can be made byfiltration and washing. When the sorbant is in a column, the separationmay take place by elution of the uncomplexed aqueous medium, again,preferably, followed by a washing step.

When the purified protein is desired free from the affinity sorbant, itcan typically be obtained by a variety of procedures. In any of thoseprocedures, the reversible linked receptor-ligand complex is dissociatedinto its component parts of support-linked receptor and oncoproteinligand, followed by separating that ligand from the linkedreceptor-ligand complex is dissociated into its component parts ofsupport-linked receptor and oncoprotein ligand, followed by separatingthat ligand from the linked-receptor to provide the purified oncoproteinfree from the affinity sorbant.

The dissociation of the reversible complex may be effected in a numberof ways. A 0.2 molar glycine hydrochloride solution at a pH value ofabout 2.5 is typically utilized. Alternatively, the bound ligand can becompeted away from the linked receptor by admixture of the reversiblecomplex with an excess of the immunogenic polypeptide utilized to raisethe receptor. Such a competition avoids possible denaturation of theligand. Separation of the admixed with the affinity sorbant. Thatadmixture forms a reversible, linked receptor-ligand complex between thelinked receptor and the oncoprotein ligand.

The ligand receptor-ligand complex is then separated from the remainderof the uncomplexed aqueous composition to thereby obtain the oncoproteinin purified form linked to the affinity sorbant. When the admixturetakes place in a beaker or flask, this separation can be made byfiltration and washing. When the sorbant is in a column, the separationmay take place by elution of the uncomplexed aqueous medium, again,preferably, followed by a washing step.

When the purified protein is desired free from the affinity sorbant, itcan typically be obtained by a variety of procedures. In any of thoseprocedures, the reversible linked receptor-ligand complex is dissociatedinto its component parts of support-linked receptor and oncoproteinligand, followed by separating that ligand from the linkedreceptor-ligand complex is dissociated into its component parts ofsupport-linked receptor and oncoprotein ligand, followed by separatingthat ligand from the linked-receptor to provide the purified oncoproteinfree from the affinity sorbant.

The dissociation of the reversible complex may be effected in a numberof ways. A 0.2 molar glycine hydrochloride solution at a pH value ofabout 2.5 is typically utilized. Alternatively, the bound ligand can becompeted away from the linked receptor by admixture of the reversiblecomplex with an excess of the immunogenic polypeptide utilized to raisethe receptor. Such a competition avoids possible denaturation of theligand. Separation of the dissociated oncoprotein ligand from theaffinity sorbant may be obtained as above.

The preparation of affinity sorbants and their use is broadly old.However, such materials and uses that incorporate the receptor moleculesof this invention have not been heretofore available. A detaileddescription of affinity sorbants, their methods of preparation and usewherein the antigen is linked to the support may be found in Antibody asa Tool, Marchalonis and Warr eds., John Wiley & Sons, New York, pages64-67 and 76-96 (1982).

VII. PANEL ASSAY

Panels of antibodies can be used to characterize virtually anybiological sample by comparing the combination of antibody/antigencomplexes formed in an unknown sample with combinations obtained from aknown body sample. Tissues, urine or other body fluids may becharacterized with respect to expression of oncogene andoncogene-related sequences. This expression is thus interpreted toindicate the stage of development of tissues or embryos or the presenceor severity of cancers.

A panel assay involves screening a sample with more than one antibody.The sample is allowed to react with each antibody, and complexes betweenthe antibodies and ligands found in the sample are detected. The patternof complexes, that is, the combination of antibodies which react withligands in the unknown, is compared to a pattern of reacting antibodiesin a known sample. The more similar the combinations are, i.e., thehigher the coincidence of the same antibody reacting with the sameligands in both the known and unknown sample, the more similar thesamples are, i.e., the higher the likelihood that the unknown sample isat the same stage of development or has the same diseased state as theknown sample.

The assay can be performed in liquid, or by use of a solid substrate, asnoted supra. Herein, aliquots biological body samples wereelectrophoresed into a gel and transferred onto nitrocellulose which wasthen cut into strips. This Western-blot approach allows the same sampleto be probed with a panel antibodies. By recreating the original blockof nitrocellulose, the identity of specific and non-specific bands canbe easily determined. For cell or tissue extracts, approximately 0.5 mgof protein was loaded per gel. For urine samples, the equivalent of 12ml of urine was added and for other bodily fluids (serum, plasma,amniotic fluid, follicular fluid, ascites fluid, saliva) 100 ul wasloaded. In addition to probing each sample with several antibodies, upto 24 samples were probed with the same antibody. This approach producesdifferential binding activities due to different antigen concentrationsand not variability due to secondary reagents or incubation conditions.The binding can also be semi-quantitated by probing samples which havebeen serially diluted to obtain relative increases in concentration overdetectable levels.

Use of the solid support provides means for analyzing samples usingautomated scanners. These scanners can be programmed to digitizeinformation on the nitrocellulose, and further programmed to comparesamples. In this way, panel assays using a multiplicity of antibodiescan be performed automatically. This can be used, for example, forcharacterizing large numbers of known samples to determine commoncharacteristics, as well as for characterizing large numbers of unknownsamples against a known. This technique also engenders more accuratescoring of reactivity patterns as such scoring is performed by use ofthe digitized information.

The sample can also be probed with a variety of antibodiessimultaneously, as by use of a cocktail of antibodies. This approach mayprovide a more efficient means of generating patterns of reactivity, butmay generate complex patterns which may be difficult to analyze.

Herein, the separated strips of nitrocellulose, each containing a laneinto which an aliquot of the sample was run, were separately probedusing an immunoblot assay as described supra.

Preliminarily, samples of known origin were probed in order to obtainprofiles against which unknown samples could be compared. Variousapproaches were used to derive such profiles. For one approach, severalantibodies which recognize the same protein were identified. Thisapproach increases accuracy of the probe identification. A secondapproach was to profile different tumor extracts with the same panel ofantibodies. This generated a variety of patterns against which unknownsamples could be compared. A third approach was to profile normaltissues to develop developmental profiles for different organs. Allthese approaches were taken in order to generate patterns of reactivityfor known samples against which patterns of unknown samples can becompared.

An example of using hybridomas with different specificities is providedby fes antibodies which produce a variety of reactivity patterns againstthe ATP binding domain of kinase genes. The similar patterns of crossactivity allow the antibodies to be grouped and tested on variousbiological specimens. In FIG. 25, a cell line containing the fesoncogene product was probed with a group of antibodies directed againsttwo regions of the fes sequence as well as another oncogene, erb B. Inlane I, the fes gene product was readily detected by an antibodydirected against a fes sequence located in another conserved kinaseregion. Over-exposure of the gel does show binding for the fes geneproduct by the antibodies used in lane A-H but the binding activity ismarkedly less than the antibody used in Lane I. As another control, thetwo antibodies respectively in lanes J and K recognized an erb B relatedprotein.

In FIG. 26, the same antibodies were simultaneously used to probe anextract from a cell line which contains the EGF receptor (which isencoded by the erb B protooncogene). This protein is readily detected bythe erb B antibodies in addition to the p130^(erb) which was also seenin the fes transformed cell line (FIG. 25). The fes antibodies used inlanes D, F, G, detect an additional protein, p30^(fes), which was notdetected in lane E. An ELISA assay showed that the cross reactivitypattern for this antibody, however, is similar to the antibody used inlane D and identical to the antibodies used in lanes F and G.

These same antibodies were used in FIG. 27 to probe a concentrated urinesample from a pregnant diabetic patient. A protein of approximately 70kdwas detected by the three antibodies used in lanes A-C while a proteinof 55 kd was detected in lanes D-H. Thus, the cross reactivity patternsof these fes proteins are useful for identifying antibodies which arelikely to recognize the same protein. The subtle differences inreactivity patterns can be used to demonstrate the presence of severaloncogene-related determinants on a single protein. This is shown by the5 antibodies which detect the 55 kd protein and recognize at least 3different fes-related epitopes. The ability of the antibodies to detectp70^(fes), p55^(fes), and p30^(fes) may also reflect onpost-translational modification differences in conformation of the ATPbinding domain of the fes oncogene product.

Profiling tumor extracts such as those derived from cell lines ondeposit at the NIH depository also provides targets for antibodyrecognition. In FIGS. 28-31, various tumor extracts were probed with fesor erb B antibodies. Lanes A-D were probed with antibodies directedagainst a portion of the ATP binding site of a kinase gene. Lanes E-Hwere probed with antibodies directed against different portions of thesame binding site. Lanes I and J are probed with antibodies directedagainst the amino end of v-erb B (173-1C11 and 173-4All) while lanes Kand L were probed with antibodies directed against a different kinasedomain. These probings required a minimal amount of material (0.5 g oftissue was probed with 64 antibodies) and produced a broad spectrum ofreactivity patterns.

In FIG. 28, an endometrial cancer extract is shown to have low levels ofp60^(fes) (lanes A-D) and p70 (lanes E-H) while p200^(erbB) (lanes I,J)is readily detected. The p200^(erbB) had previously been detected athighest levels in embryonic heart (data not shown). This expression ofp220^(erbB) appears to be inappropriate with respect to time and tissue.

In FIG. 29, a metastatic breast tumor extract was probed with the sameantibodies. In this tumor, p70^(fes) is readily detected in the absenceof p60^(fes). The antibodies directed against the amino end of erb Bdetect p30^(erbB), and p35^(erbB), and p40^(erbB) while the antibodiesdirected against the carboxyl portion of the viral protein detectp130^(erbB).

In FIG. 30, another breast cancer extract produced another reactivitypattern. In addition to p70^(fes), a weak activity for p80^(fes) wasdetected. Both of these proteins were detected with all four antibodiesalthough this group recognized at least three different epitopes. An erbB doublet, p130^(erbB), was also weakly detected (lanes K,L).

In FIG. 31, an ovarian carcinoma produced another reactivity pattern.p60^(fes) was weakly detected (lanes A-D) as was p35^(fes) (lanes E-H)while p70^(fes) was readily detected (lanes A-D) as was p35^(fesB)(lanes I,J). Thus, each tumor displayed a unique reactivity pattern andeach protein described was detected by at least 2 antibodies. Theproteins in lanes E-H contained at least three res-related epitopesalthough the proteins were probably not encoded by the fes oncogene.

In FIGS. 32-39 several additional oncogene-related proteins weredetected with another panel of antibodies. In lane A of each Figure, anantibody to the ros sequence of a conserved kinase gene was used whilean antibody to a different kinase domain III was used in lane B. InLanes C-G, 5 antibodies to the amino end of β TGF were used as probes.Three antibodies to the unique carboxyl portion of the H-ras sequencewere used in lanes H-J while four antibodies to a conserved region inthe hormone binding domain of erb A were used in lanes K-N.

In FIG. 32, a breast carcinoma extract was probed with this series ofantibodies. In lanes A and B, a 150 kd protein was detected with ros andfes antibodies directed against two different conserved regions of thekinase domain suggesting extensive kinase homology. The ros antibodiesalso detected a protein of 120 and 40 kd. In lanes C-G, a 25 kd proteinwas detected by all β TGF antibodies. These antibodies recognize atleast 3 different epitopes based upon immunoblots (note the additionalband at 45 kd in lane C). In lanes H-J, five different ras-relatedproteins were detected. The ratios of binding activities for theseras-related proteins suggests each of these antibodies detects a uniquedeterminant. The antibody (146-3E4) in lane H preferentially detectedp200^(ras), p48^(ras), and p27^(ras). In contrast, the antibody used inlane J (146-l7A5) preferentially detects p2las. In this exposure, thesedifferences were most readily seen by comparing the intensities ofp27^(ras) with p21^(ras). Although p27 was only weakly detected in laneJ and p21 was only weakly detected in lane H, over-exposure of the gelindicates all 5 ras-related proteins were detected by all threeantibodies.

The ovarian carcinoma probed in FIG. 33 is similar to FIG. 8 exceptpl50ras/fes was not detected but a 50 kd β TGF protein appears in laneG. The metastatic colon carcinoma probed in FIG. 34 is also similarexcept the 45 and 50 kd β TGF proteins are not detected. The ovarianextract in FIG. 35 is also similar except p45^(ras) and p52^(fes) werepresent at higher concentrations relative to the other ros-relatedproteins and the ros-related proteins were not seen.

The lymphoma extract probed in FIG. 36 is unique due to the relativelyhigh concentration of p27. The breast carcinoma extract probed in FIG.37 produced another unique reactivity pattern. Although little activitywas detected for the ros, fes, or ras-related proteins, p24 β TGF wasreadily detected is in p22^(erbA) and p55^(erbA). p22^(erbA) wasrecognized by all four antibodies while p55^(erbA) was readily detectedonly by the antibody used in lane N. Overexposure showed binding forthis protein by the other three erb A antibodies. The differentialactivity with p55^(erbA) and p22^(erbA) indicate both of these proteinshave two erb A related epitopes. Moreover, the antibodies did not crossreact with the homologous region of the glucocorticoid or estrogenreceptor and the antibodies produce nuclear staining patterns (data notshown). Thus, in contrast to the extracts probed in FIGS. 32-37, thebreast tumor has elevated levels of p22^(erbA). The p22^(erbA) proteinwas also seen in the rectal tumor extract of FIG. 38 although p55^(erbA)was not detected even in the overexposed gel. In FIG. 39, the metastaticlung extract had p52^(fes) and a unique activity pattern for the rasprobes. Although p48^(ras) and p200^(ras) were readily detected, therewas very little activity seen for p27^(ras) or p21^(ras). This resultwas similar to the profile of several embryonic tissues (see below). Asthese tissues develop, the p200^(ras) concentration decreased while thep27^(ras) concentration increased.

Although considerable diversity is seen in the tumor samples, remarkablesimilarities were found in normal tissues. These similarities allowdevelopmental profiles for different organs to be generated. In FIG. 40,a develop-mental profile of rat striatum was produced with 7anti-bodies. In lane A, p21^(ras) and p25^(ras) were detected in the 18day old embryo. By day 2, pl50^(ras) was barely detected in addition top2l^(ras) and p25^(ras) was readily detected and is present insubsequent panels. In lane B, p52^(ras) was readily seen at 18 days offetal development. Overexposure of the gel revealed p200^(ras). By 18days, p27^(ras) was easily seen (p200^(ras) was no longer detectable inoverexposed gel). The concentration of p27^(ras) was highest by day 70.p2l^(ras) was also readily detectable at day 70 although overexposureindicated p21^(ras) was present at all five time points. In lane C,pl50^(myc) was also an adult specific protein which was barely detectedat day 2 but readily seen by day 18. In lane D, pl20^(myb) was an embryospecific protein which was detected only in the fetal panel. In lane E,p100^(int-1), p70^(int-1), p45^(int-1), p90^(sis), and p56^(sis) do notappear to be developmentally regulated. In lane G, p60^(sis) was highestin the embryo panel. Thus, in contrast to the tumor extracts describedin FIGS. 4-15, the oncogene-related protein profiles of normal tissuesare much more uniform and tightly regulated.

Thus, FIGS. 25 through 40 show that a known sample can be probed withvarious antibodies in order to generate a pattern of reactivity. Theabove examples show the cross reactivity of various antibodies not onlyamong the same proteins in different samples but also among variousproteins.

A. Classification of Known Samples

Tumor extracts or other body samples may also be characterized accordingto the various gene products produced. Presently, various tumor extractx antibody combinations were scored using the immunoblot technique todetermine the presence and levels of various oncogene products. Thesedata are presented at FIGS. 41 and 42. Tumor extracts were derived fromcell lines on deposit at the NIH depository, and samples wereelectrophoresed and blotted onto nitrocellulose as described above.Antibodies against polypeptides encoded by oncogenes from variousoncogene families were used to probe the samples.

The reactivities were scored for the presence and level of expression ofoncogene product. In FIG. 41, p52^(ras) was detected in all of theovarian extracts but highest levels were found in tumors listed asminimum or moderate. p60^(src) and p48^(src) were predominantly detectedin the ovarian tumors with highest frequencies in the advanced category.p125^(ros) and p15^(ros) were detected in most of the tumors but in thebreast extracts, expression was concentrated in the adenomas and thosecarcinomas classified as minimal or moderate. p150^(ros) expression wasconcentrated in the breast tumors in contrast of p120^(ros) which wasfound in only half of the breast tumors although this protein wasdetected in most other tumors with the exception of the endometrialextracts. p22^(erb) A was scattered throughout these tissues but was notfound in any of the extracts listed as advanced.

Additional segregation of activities are seen in FIG. 42 which listsseveral of the kinase-related proteins. In this table, the high levelsof p70^(fes) in the ovarian and endometrial extracts is striking.Similarly, the restriction of p130^(erb) B to the ovarian and lungextracts may be significant.

Thus, tumor extracts can be characterized with respect to oncogene oroncogene-related products. Using specific antibodies, proteins common tospecific tumor types can be detected. These patterns of reactivity canbe used as a standard against which patterns of unknown samples may becompared.

B. Markers in Body Fluids

The panel assay may also be used to monitor protein production in asingle individual. In this way, the effects of therapy may benon-invasively monitored by screening, for example, urine samples. Thisapproach involves the screening of an original sample from a patient inorder to obtain a profile of the activity pattern of a panel ofantibodies. As therapy progresses, subsequent samples are monitoredusing the same original sample as a standard for comparison. FIG. 43shows sequential urine samples from gestational trophoblast diseasepatients undergoing chemotherapy. The asynchronous appearance of anumber of oncogene-related proteins is apparent. These data can befurther correlated with clinical data regarding therapy effectiveness inorder to monitor patients after therapy.

C. Detection of Oncogene-Related Proteins

Antibodies against polypeptides encoded by oncogenes may recognize otherproteins which also contain the conserved polypeptide or portionsthereof. Thus, panels of antibodies may be used to detectoncogene-related proteins in a sample by probing the sample withmultiple antibodies each against distinct regions of the polypeptide. Bydetermining a pattern of reactivity with different antibodies, differentoncogene related proteins can be identified.

The p21^(ras) detected by the H-ras-specific antibody represents asubset of the p21^(ras) detected by the broadly reactive antibody. Allsamples containing p21 detected by the H ras specific antibodiescontained p21 detected by antibody 142-24E05 but not vice versa. Theother proteins detected by the H ras specific antibodies were notdetected by 142-24E05 indicating the conserved ras region is absent orat least altered so as to preclude 142-24E5 binding. However, theconcordance of binding by the three H-ras specific antibodies indicatesthe same protein was detected by all three antibodies. The similar ratioof binding of the antibodies to p200^(ras), p48^(ras), and p27^(ras)suggests some structural similarity in the epitopes detected by thethree antibodies. This similarity can be addressed using a number oftechniques. One approach is to digest labelled antigens and subject thedigest to two dimensional peptide mapping. If the larger molecule is notwell recognized in solution, additional approaches are possible. Forexample, samples containing different combinations of immunologicallyrelated proteins could be subjected to partial digestion andimmunoblotted. If there is a precursor-product relationship, thesmallest size protein detected with the antibody should be the same (acontrol incubation without added proteases would control for sampledifferences of endogenous proteases). Alternatively, the partial digest(to expose the antigenic site) may be immunoaffinity purified and thenimmunoblotted or peptide mapped. Identity of similar sized proteinsdetected by different antibodies or the same antibody in differentsamples (i.e. urine and tissue) may be tested using similar approachesor the isoelectric point could be determined using two dimensional gelsand immunoblot detection. This approach is also useful foridentification of multiple forms migrating at similar rates on SDSpolyacrylamide gels.

VIII. SERUM SCREENING IN ASYMPTOMATIC INDIVIDUALS

Set forth below are studies on two different cohorts of workers known tohave exposure to environmental carcinogens. None of the workers showedclinical symptoms of neoplastic disease. Both studies show that theindividuals with the highest exposure to carcinogens also show abnormalexpression of oncoprotein. In the first example, one individual withknown exposure to several carcinogens, including PCB's, asbestos, andcigarette smoke also showed a fifty-fold elevated level of H-ras protein21. Eighteen months after this screening, this individual developed acolorectal adenoma. Upon removal of the adenoma, the serum levels ofH-ras p21 returned to normal. In the second example, foundry workersexposed to polyclic aromatic hydrocarbon carcinogens were found to haveabnormally elevated expression of the fes oncogene-related protein. Theabnormal expression was found only in some of the workers with thehighest workplace exposures to the carcinogen. Unexposed individuals hadnon-detectable levels of oncoprotein. These studies show the utility ofthe present methods and monoclonal antibodies for cancer prognosis, andalso show important markers which indicate exposure to environmentalcarcinogens. The H-ras p21 marker is unexpectedly useful for prognosisof colorectal adenomas. The fes oncogene-related protein is useful as anindicant of polyclic aromatic hydrocarbon exposure.

EXAMPLE 1 Serum Screening of Workers Exposed to PCBs

Serum from a group of sixteen municipal workers who had possibleprevious exposure to polychlorinated biphenyls (PCBs) was screened usingmonoclonal antibodies raised against polypeptides encoded by oncogenes.The results presented herein show that the individual with the worsthistory of exposure to carcinogens also showed the most abnormalexpression of H-ras p21. Eighteen months after the serum screening, thisindividual developed a colorectal adenoma. Upon surgical removal of theadenoma, expression of H-ras reverted to normal.

Methods

1. Individual Background Screen

Sixteen municipal workers who had been involved in the clean up of PCBcontaining transformers were evaluated for medical and occupationalhistory, as well as examined physically and clinically. The transformeroil contained approximately 600,000 ppm PCBs, and the clean-up lastedseveral months. All subjects were white males with an average age of 42,and the range of ages from 27-65.

The workers were questioned regarding exposure to PCBs from othersources, as well as exposure to other carcinogens, such as asbestos andcigarette smoke, and examined physically, with particular reference todermatologic examination, laboratory tests were performed for analysisof serum triglycerides, serum PCB levels, and liver function tests(SGOT, SGPT, LDH, alkaline phosphatase, and bilirubin; subjects wererequested to refrain from ethanol consumption for two weeks prior totesting, and to have no oral intake on the day of testing). Tests werecarried out by routine methods.

All sixteen subjects reported some dermal contact with thePCB-containing transformer oil during this work period; in addition, twohad prior potential exposure to PCB-containing transformer oil over thepast twenty years (patients 9 and 10). Seven workers reported havingworked with other carcinogenic materials in the past; this was primarilyasbestos exposure from insulation handling, although one individual alsohad significant work exposure to chlorinated hydrocarbon solvents andionizing radiation (patient 14). Twelve of the workers were currentcigarette smokers or recent ex-smokers (within the past 5 years) andfour had never smoked. Two individuals reported having acneiform lesionson the arms, legs and feet shortly after exposure (patients 4 and 8),but physical examination revealed no abnormalities consistent with PCBexposure in any of the workers.

In all cases, liver function tests were within normal limits. In onlythree cases were serum triglycerides elevated. One individual (patient8) had a very high serum triglyceride level which had been reportedlynormal in the past prior to PCB exposure; however, his serum PCB levelwas quite low. In the other two hypertriglyeridimic individuals, theprior status in terms of triglyceride levels was unknown, and theirserum PCB levels were also relatively low. Furthermore, the individualwith the highest serum PCB level had a normal triglyceride level. On thebasis of these data, it is impossible to draw only conclusions regardingcorrelations between relatively low serum PCB levels and serumtriglycerides, an association which has been noted with higher PCBexposures.

As noted, overall serum PCB levels were quite low, attesting to theadequacy of protective measures taken. In general, levels less than 10ppb are not viewed with concern since such levels can often beidentified in non-occupationally exposed normal controls. But thismeasure, only one individual, patient 6, could be considered to have aseriously elevated serum PCB level.

Patient 6, the individual shown to have extremely aberrant rasexpression, also had the highest incident of known carcinogen exposure.Patient 6 was a 57 year old white male. At the time of the serumscreening, he showed no overt symptoms of neoplastic disease. Theindividual had worked in building maintenance for 25 years, a job whichincluded the spraying and removal of asbestos insulation, theapplication of various pesticides including chlordane, and the clean-upof transformer oils containing PCBs. He had also smoked a pack ofcigarettes a day for many years. His physical examination was notableonly for mild hypertension.

2. Serum Screening

An adaptation of the urine immunoblotting technique of Niman et al.,PNAS-USA 82:7924-7928 (1985); herein incorporated by reference, was usedfor screening serum. For the assay, subject serum was prepared andprobed with monoclonal antibodies raised against polypeptides asdescribed infra. The monoclonals used are designated infra.

For the assay, 100 μl of serum was mixed with 400 phosphate-bufferedsaline (PBS) at pH 7.4, 25 μl 2-mercaptoethanol and 475 μl of samplebuffer in deionized water (6.25% sodium dodecyl sulfate, 6.25%glycerol), and placed in a boiling water bath for 5 minutes. Sampleswere then loaded on a 5-17% polyacrylamide gel and electrophoreticallyseparated and transferred to nitrocellulose. After blocking with PBScontaining 3% bovine serum albumin and 0.1% Triton X-100, thenitrocellulose was incubated overnight at 4° C. with monoclonalantibodies directed against synthetic peptides representing predictedoncogene sequences (ascites fluid diluted 1:2000). After washing threetimes, the nitrocellulose was incubated with rabbit anti-mouse IgF(1:500) for 60 minutes at room temperature. After three more washes, thenitrocellulose was incubated with ¹²⁵ I-labelled protein A (10⁶ cpm/ml).Binding was visualized with intensifying screens.

The primary antibodies utilized were directed against protein sequencesof the following oncogenes (see FIGS. 44(A) and (B)):

sis (lane 1, hybrid 112-09Bl0 (ATCC HB8800), sequence SLGSLTIAEPAMIAEC);

fes (lane 2, hybrid 127-42C11 (ATCC HB9561)and lane 3, hybrid 127-50D04(ATCC HB8968), sequence LMEQCWAYEPGQRPSF; lane 15, hybrid 121-l4C09(ATCC HB9875), sequence IGRGNFGEVFSG(C));

β-TGF (lane 4, hybrid 100-30C05 (ATCC HB9787) and lane 5, hybrid100-34E06 (ATCC HB9788), sequence ALDTNYCFSSTEKNC); int-1 (lane 6,hybrid 222-35C08 (ATCC HB9052) and lane 12, hybrid 222-37F04 (ATCCHB9786), sequence LHNNEAGRTTVFS(C));

myb (lane 7, hybrid 133-l0F06 (ATCC HB9077), sequence LGEHHCTPSPPVDHG);

src (lane 8, hybrid 203-07Dl0 (ATCC HB8898), sequence(C)GSSKSKPKDPSQRRHS);

c-myc (lane 9, hybrid 155-11C07 (ATCC HB8976), lane 13, hybrid 155-08G01(ATCC HB9001), and lane 14, hybrid 155-09F06 (ATCC HB9000), sequenceCSTSSLYLQDLSAAASEC);

mos (lane 10, hybrid 165-35F02 (ATCC HB9784), sequence LGSGGFGSVYKA(C));

H-ras (lane 11, hybrid 142-24E05 (ATCC HB8679), sequenceYREQIKRVKDSDDVPMVLVGNKC and lane 16, hybrid 146-03E04 (ATCC HB8997),sequence YTLVREIRQHKLRKLNPPDESGPGC).

Results and Discussion

Results are presented in Table 7, below. The most striking result isthat for patient 6. Patient 6 had the worst exposure to carcinogens ofthe 16 workers tested. This individual showed the highest serum PCBlevels, and was also positive for asbestos exposure and smoking. As canbe seen Table 7, this individual showed abnormal expression of both fesand ras markers, with the ras marker expression being far above thenormal range. In a follow-up study, set forth infra, this individual wasshown to have developed cancer; upon surgical removal of the canceroustissue, ras expression reverted to normal.

The findings with regard to other workers also show several patterns ofexpression. One-half of the smokers (patients 3-9) showed abnormalfes-oncogene related banding patterns, whereas none of the non-smokersdisplayed this pattern. One smoker also had an aberrant pattern for sisoncogene-related proteins, but this is of unknown significance. It is ofnote that example 2, infra, also shows abnormal expression of fesoncogene-related protein only in individuals known to be exposed topolycyclic aromatic hydrocarbon carcinogens, and these individuals werealso smokers.

The patterns for the other oncogene or oncogene-related proteins werenormal except for ras. Apart from patient 6, two other individuals haddetectable levels of ras p21. In both of these cases, the bands wererelatively weak, and there did not appear to be any connection toexposure to PCBs. One patient (10) had a PCB level of 6.5 with apositive smoking history and a positive history of other carcinogenexposure (asbestos). The other individual (patient 11) had a PCB levelof 0.3 and no other known exposure history.

                                      TABLE 7                                     __________________________________________________________________________    Summary of Findings on PCB Exposed Workers                                    PCB Levels (ppb)                                                              2,2'3,3',                                                                              2,2'3,3',                                                                         2,2'3,4,                                                                          2,2'3,4,                                                                          2,2',4,                                                                           2,3,3',                                                                           2,3,4,                                                                            2,3',                                                                             Liver                                                                            Trigly-                                                                           History                                                                            Other Serum Oncogene         4,4'5    4,4'5,5'                                                                          4'5'                                                                              4'5 4'5,5'                                                                            4'4 4'5 4,4'                                                                              Func-                                                                            cerides                                                                           of   Carcinogen                                                                          Proteins               Patient                                                                            Hepta                                                                             Hepta                                                                             Hexa                                                                              Hexa                                                                              Hexa                                                                              Penta                                                                             Penta                                                                             Tetra                                                                             tion                                                                             (mg/dl)                                                                           Smoking                                                                            Exposure                                                                            fes                                                                             ras                                                                              sis               __________________________________________________________________________    1 1.0                                                                              --  --  0.5 --  0.5 --  --  --  WNL                                                                              WNL Yes  No    - -  -                 2 2.2                                                                              --  --  0.8 --  0.8 --  0.6 --  WNL                                                                              WNL Yes  No    - -  -                 3 0.6                                                                              --  --  0.6 --  --  --  --  --  WNL                                                                              WNL Yes  No    ++                                                                              -  -                 4 2.8                                                                              --  1.3 0.8 --  0.7 --  --  --  WNL                                                                              193 Yes  Yes   ++                                                                              -  ++                5 6.3                                                                              --  1.9 1.5 --  1.7 --  1.2 --  WNL                                                                              WNL No   No    ++                                                                              -  -                 6 22.2                                                                             0.6 2.0 5.2 0.4 3.6 2.5 7.2 0.7 WNL                                                                              WNL Yes  Yes   ++                                                                              +++                                                                              -                 7 0.4                                                                              --  --  --  --  0.4 --  --  --  WNL                                                                              WNL Yes  No    ++                                                                              -  -                 8 1.8                                                                              --  0.4 0.6 --  0.8 --  --  --  WNL                                                                              597 Yes  No    ++                                                                              -  -                 9 3.0                                                                              --  2.1 0.3 --  0.6 --  --  --  WNL                                                                              271 Yes  Yes   ++                                                                              -  -                 10                                                                              6.5                                                                              --  2.3 1.6 --  1.8 --  0.8 --  WNL                                                                              WNL Yes  Yes   - +  -                 11                                                                              0.3                                                                              --  0.3 --  --  --  --  --  --  WNL                                                                              WNL No   No    - +  -                 12                                                                              0.3                                                                              --  --  --  --  0.3 --  --  --  WNL                                                                              WNL Yes  Yes   - -  -                 13                                                                              2.0                                                                              --  0.3 0.6 0.4 0.7 --  --  --  WNL                                                                              WNL No   Yes   - -  -                 14                                                                              1.5                                                                              --  --  0.6 0.4 0.5 --  --  --  WNL                                                                              WNL No   Yes   - -  -                 15                                                                              4.5                                                                              --  0.5 1.1 0.3 1.6 0.4 0.6 --  WNL                                                                              WNL Yes  No    - -  -                 16                                                                              0.9                                                                              --  --  0.9 --  --  --  --  --  WNL                                                                              WNL Yes  No    - -  -                 __________________________________________________________________________     WNL  within normal limits; - = negative; + = weakly positive; ++ =            moderately positive; +++ = strongly positive                             

Patient 6 was extremely aberrant for H-ras p21 expression. At lanes 11and 16 of FIG. 44B, the bands for p21 are shown to be quite pronounced.Cf., lanes 11 and 16 of FIG. 44A which show no p21 expression fornormal, unexposed individuals. It is worth noting that the identity ofp21 was confirmed by use of antibodies directed to the conserved regionof ras p21 (lane 11) as well as the H-ras specific carboxy terminalregion (lane 16), and the use of antibody or other receptors directed toboth conserved regions as well as protein-specific regions is a methodgenerally applicable to confirm the identity of a protein marker, if itis desired. The identity of the protein, however, may not be criticalwhere the important information derived is the relative presence orabsence of the protein, rather than the composition of the proteinitself. Thus, patient 6, the individual with the most extreme exposureto known carcinogenic substances also was found to be the individualmost aberrantly expressing H-ras p21.

These data show that ras oncogene p21 is a useful as a marker forscreening to determine if an individual has been exposed to acarcinogen. The follow-up data, presented below, show that this methodand the receptors used are useful tools for cancer prognosis.

Follow-Up Study On Patient 6

Approximately 18 months after the serum screening which showed highlyelevated levels of ras protein, patient 6 manifested clinical symptomsof cancer. Patient 6 developed rectal bleeding, and colonoscopy revealeda 2 cm. tubulo-villous adenoma of the descending colon. This adenoma wasremoved surgically. Patient 6 was re-screened approximately 6 weeksafter removal of the adenoma. This screen showed expression of the rasoncogene proteins had reverted to a normal pattern. These patterns areshown at FIG. 45. Lane A shows ras p21 expression 18 months prior to theshowing of clinical symptoms. Lane B shows that, 6 months after theremoval of the adenoma, the ras band is not visible.

These data show that H-ras p21 expression can be correlated todevelopment of colonic carcinomas as well as exposure to carcinogens.Detection of this abnormal H-ras expression some 18 months prior to thedevelopment of clinical symptoms of cancer shows the value of thismarker as a prognostic tool for cancer.

To what specific carcinogen or mixture of carcinogens the colorectalcarcinoma can be attributed is unclear. Patient 6 did not have anydocumented exposure to the carcinogens known to activate the ras gene,except for benzo(a) pyrene from cigarette smoking. See Brandt-Rauf andPincus, Occup. Med. 2:27-38(1987) and Spandidos and Kerr, Br. J. Cancer49:681-688 (1984), both of which are incorporated herein by reference,for a discussion of carcinogens which activate ras (benzo(a)pyrenes,dimethylbenzathracene, N-nitroso compounds and ionizing radiation) andthe expression of ras in human colonic carcinomas and pre-malignantcolonic polyps. Patient 6 did have a long history of exposure toasbestos, which has been implicated in the development of colo-rectalcancer. Wylie et al., Sem. Occupl. Med. 2:291-309 (1987). Whetherasbestos, by itself or in combination with other environmentalcarcinogens, activated the ras oncogene, is not clear. It is clear,however, that (a) H-ras p21, with its expression well in advance of themanifestation of clinical symptoms of neoplastic disease, is a powerfulprognostic tool, (b) the methods presently described provide for anaccurate and non-invasive screening, the results of which are useful topredict the onset of clinical symptoms of neoplastic disease, and (c)the receptors herein described, i.e., those directed against H-ras p21,whether in the form of antibodies or fragments thereof or nucleic acids,are useful to detect these prognostic markers.

One explanation for the overexpression of p21 by patient 6 may be thatexposure to asbestos caused chromosomal abnormalities which led to theactivation of the ras gene. While in the present example the specificpathogenic machinery is not clearly understood, other oncogenes, forwhom environmental activators are known, may be chosen as markers forthe present screen. It is possible, however, that the clastogenic effectof asbestos could manifest itself in the activation of the ras gene.See, Jaurand et al., Mut. Res. 169:141-148 (1986), and Kelsey et al.,Br. J. Cancer 54:107-114 (1986) where asbestos fibers are shown to beclastogenic on cells in culture. For example, the myc oncogene has beenshown to be activated as a result of translocation of the proto-oncogeneto a site where transcription is accelerated, Erikson, et al., PNAS-USA80: 820-824 (1983), and a similar scenario may be envisioned forasbestos-caused ras activation. More recent studies have shown thatasbestos fibers are capable of transfecting exogenous DNA segments,including oncogenes and promoter sequences, into primate cells inculture and producing cell transformation. Appel, et al., PNAS-USA 85:7670-7674 (1988), and this may be the mechanism of ras activation.

It is known that the introduction of the ras proto-oncogene linked to aviral transcriptional promoter into cells in culture results inincreased expression of the gene and causes malignant transformation ofthe transfected cells. Chang et al., Nature 297: 479-483 (1982). Thus,in this individual, it is possible that prolonged exposure to asbestosfibers produced increased expression of the ras proto-oncogene in hiscolonic epithelium. This was manifested by increased quantities of theproto-oncogene encoded p21 protein in his serum. Ultimately, theproto-oncogene over-expression led to colonic neoplasia, in this case, atubulo-villous adenoma which was identified clinically 18 months priorto its progression to a malignant growth. When the adenoma wassurgically removed, the source of the ras encoded p21 protein wasremoved, and p21 protein was no longer detected in the patient's serum.

Thus, while the present study was based on a group of individuals whohad all been exposed to a known environmental carcinogen, PCB, theresults unexpectedly yielded a marker which may be useful in predictingneoplastic disease resulting from asbestos exposure. In any event, thepresent study shows that the present method is useful for predicting theonset of cancer prior to manifestation of clinical symptoms.

EXAMPLE 2 Serum Oncogene Proteins In Foundry Workers

In this study, a well-defined occupational cohort of iron foundryworkers with known, quantified exposure to a common occupationalcarcinogen, benzo(a)pyrene were assayed to determine if any oncogene oroncogene-related markers are associated with exposure to thiscarcinogen. Benzo(a)pyrene (BP)and related polycyclic aromatichydrocarbons (PAHs)have been associated with increased risk of lungcancer in smokers, coke oven workers and foundry workers (Redmond etal., Annals of the New York Academy of Sciences 271:12 (1976); IARC,Polynuclear aromatic compounds, In: Monographs on the Evaluation of theCarcinogenic Risk of Chemicals to Humans, Vol. 34, Part 3, Lyon: IARC(1984); IARC, Tobacco smoking, In: Monographs on the Evaluation ofCarcinogenic Risk of Chemicals to Humans, Vol. 38, Lyon: IARC (1986).Such PAHs have been shown to be capable of activating oncogenes both invivo and in vitro. Ballmain and Pragnell, Nature 303:72 (1983); Marshallet al., Nature 310:586 (1984). Furthermore, not only are extensiveoccupational histories available for this cohort, but also theirexposure has been well-defined in terms of workplace air levels of BP. Aclear dose-related increase was seen in levels of PAH-DNA adducts in theperipheral leukocytes of these workers , Perara et al., Cancer Res.48:2288 (1988b), which was consistent with measurements of DNA adductsby the 32p postlabeling method, Phillips et al., Mutation Res. 204:531(1988); Hemminki et al., J. of Work Environment and Health 14:55 (1988).This cohort therefore represents a model population for the study ofoncogene activation related to occupational exposure.

Materials and Methods

The study cohort of workers employed at an iron foundry in Finland hasbeen previously described (Hemminki et al., 1988, supra; Perera et al.,1988b, supra). Briefly, the foundry workers were segregated by twoindustrial hygienists familiar with the workplace into groups accordingto the level of exposure to BP based on extensive industrial hygienesampling data and job description. This classification was consideredrepresentative of exposure during the past 5-10 years, since individualsin this plant tend to remain in the same job and are generally long-termemployees. Workers with 8 hour TWA exposures greater than 0.02 ug/m³were classified in the high exposure group and workers with TWAexposures between 0.05 and 0.2 ug/m³ were classified in the mediumexposure group; peak BP levels as high as 2-3 ug/m³ were known to occurfor certain individuals in the casting and shakeout areas of thefoundry. Clinical information including cigarette consumption wascollected on all workers. Unexposed controls for comparison wererecruited from patients referred to the Institute of Occupational Healthfor evaluation of possible occupational disease unrelated to PAHexposure or cancer. The control population had a lower average cigaretteconsumption than the foundry workers but was similar in terms of age andsex distribution (Perera et al., 1988b, supra).

For the purposes of this study, repeat peripheral blood samples wereavailable for 8 exposed workers (3 in the high exposure group and 5 inthe medium exposure group) and for 10 unexposed controls (a total oftwenty-eight samples). Two or three blood samples had been collected onthe workers at different times. For all workers, samples were availableimmediately following a month-long vacation and 6 weeks after returningto work. For two of the workers in the medium exposure group, anadditional blood sample was collected during the following 12 months.Single peripheral blood samples were collected on the unexposedcontrols. Blood samples (30-50 ml) were collected in heparinized plastictubes, coded and centrifuged. Buffy coat, red cells and plasma werecollected ad stored frozen at -70° C. until time of analysis. PAH-DNAadducts were determined for these same workers as reported previously(Perera et al., 1988b, supra). The values for DNA adducts in a subset ofsamples assayed for serum oncogene proteins are given in Table 8;missing values reflect the fact that inadequate amounts of DNA wereavailable for some samples.

These samples have now been analyzed blind for the presence of proteinproducts encoded by nine different oncogenes (sis, fes, B-TGF, int-1,myb, src, myc, mos, and ras) by the immunoblotting technique aspreviously described (Niman et al., 1985, supra; Brandt-Rauf and Niman,Brit J. Indus. Med. 45:689 (1988)). Briefly, 100 ul of serum is mixedwith 400 ul of phosphate-buffered saline PBS) at pH 7.4, 25 1 of2-mercaptoethanol, an 475 ul of sample buffer in deionized water (6.25%sodium dodecyl sulfate, 6.25% glycerol), and placed in a boiling waterbath for five minutes. Samples were then loaded on a 5-17%polyacrylamide gel and electrophoretically separate and transferred tonitrocellulose. After blocking with PBS containing 3% bovine serumalbumin and 0.1% Triton X-100, the nitrocellulose is incubated overnightat 4° C. with monoclonal antibodies directed against synthetic peptidesrepresenting predicted oncogene sequences (ascites fluid diluted1:2000). After exhaustive washing, the location of the oncogeneprotein-antibody bands on the nitrocellulose is determinedcolorimetrically using a secondary anti-mouse IgG antibody and anavidin-biotin-peroxidase complex (Vectastain; Vector Labs, Burlingame,Calif.). Approximate quantification of positive results is achieved byserial dilution of samples until no difference from normals isdetectable; a five-fold increase in protein expression is considered apositive result. The primary antibodies used were directed against thefollowing oncogene protein sequences:

sis (hybrid 112-09B10) (ATCC HB8800), sequence SLGSLTIAEPAMIAEC)

fes (hybrid 127-42C11) (ATCC HB9561) and 127-50D04 (ATCC HB 8968),sequence LMEQCWAYEPGQRPSF, and hybrid 121-14C09 (ATCC HB9875), sequenceIGRGNFGEVFSG(C))

B-TGF (hybrid 100-30C05 (ATCC HB9787) and hybrid 100-34E06 (ATCCHB9788), sequence ALDTNYCFSSTEKNC)

int-1 (hybrid 222-35C08 (ATCC HB9052) and hybrid 222-37F04 (ATCCHB9786), sequence LHNNEAGRTTVFS(C))

myb (hybrid 133-10F06 (ATCC HB9077), sequence LGEHHCTPSPPVDHG)

src (hybrid 203-07D10) (ATCC HB8898), sequence (C) GSSKSKPKDPSQRRHS)

myc (hybrids 155-11C07 (ATCC HB8976), 155-08G01 (ATCC HB9001), and155-09F06 (ATCC HB9000), sequence CSTSSLYLQDLSAAASEC)

mos (hybrid 165-35F02 (ATCC HB9784), sequence LGSGGFGSVYKA(C))

ras (hybrid 142-24E05 (ATCC HB8679), sequence YREQIKRVKDSDDVPMVLVGNKC,and hybrid 143-03E04 (ATCC HB8997), sequence YTLVREIRQHKLRKLNPPDESGPGC)

In this immunoblotting system, these antibodies have been found to givespecific, sensitive and reproducible results. The specificity of theantibodies has been demonstrated by the blocking of the activity bypreincubation with the specific peptide for the oncogene and the failureof the blocking of the activity with peptides of other oncogenes (Nimanet al., 1985). The assay system is capable of detecting proteins in thenanogram range and is found to give reproducible results when repeatedon the same sample.

Results and Discussion

The results, as shown in Table 8, show that two different workers(patients 2 and 7), both of whom were known to have either high (2) ormedium (7) exposure to PAHs, were positive for increased levels of fesoncogene-related protein products. The serum samples from each of theseindividuals which tested positive were drawn post-vacation and after sixweeks of work. There were no positive bands for the proteins of seven ofthe oncogenes in any of the samples (sis, B-TGF, int-1, myb, src, myc,mos). Only one sample, from patient 5, was found to be positive for rasoncogene-related protein products; however, in this case, two othersamples from the same worker, taken at different times, were negative.All results for serum oncogene proteins in the unexposed controls werenegative.

In this study it is notable that the only positive results for elevatedserum levels of oncogene-related proteins were obtained in individualsin the medium or high exposure groups. Furthermore, as shown in Table 8and reported previously (Perera et al., 1988b, supra), these individualsalso have high levels of PAH-DNA adducts compared to low exposed orunexposed individuals. Although two of the individuals with positiveoncogene proteins were also smokers of approximately one pack ofcigarettes per day (patients 2 and 5), it is likely that the majorcontribution to their body burden of PAHs was due to workplace exposure.For example, exposure at 0.2 ug/m³ is approximately equal to the BP dosefrom smoking 5 to 7 packs of cigarettes per day.

                                      TABLE 8                                     __________________________________________________________________________    Serum Oncogene Protein Expression and PAH-DNA Adducts in Foundry Workers      and Unexposed Controls                                                        Ambient          PAH-DNA Adduct Levels.sup.1,                                                                 Serum Oncogene Proteins.sup.1                 Exposure,  Smoking                                                                             fmol/ug        fes            ras                            ug BP/m.sup.3                                                                        Patient                                                                           cigarettes/d                                                                        Post-Vacation                                                                        Work 1                                                                            Work 2                                                                            Post-Vacation                                                                        Work 1                                                                            Work 2                                                                            Post-Vacation                                                                        Work                                                                              Work                __________________________________________________________________________                                                              2                   >0.2   1   0     0.11   0.8 NA  -      -   NA  -      -   NA                  (high  2   20    0.13   2.0 NA  +      +   NA  -      -   NA                  exposure)                                                                            3   0     NA     2.8 NA  -      -   NA  -      -   NA                  0.05-0.2                                                                             4   15    0.13   0.36                                                                              NA  -      -   NA  -      -   NA                  (medium                                                                              5   20-25 NA     0.32                                                                              0.42                                                                              -      -   -   -      +   -                   exposure)                                                                            6   15    NA     0.8 0.5 -      -   -   -      -   -                          7   0     0.48   1.28                                                                              NA  +      +   NA  -      -   NA                         8   20    ND     0.4 NA  -      -   NA  -      -   NA                  <0.05  9   15     0.08          -              -                              (unexposed                                                                           10  10    ND             -              -                              controls)                                                                            11  0      0.14          -              -                                     12  0     ND             -              -                                     13  0     ND             -              -                                     14  0     0.3            -              -                                     15  0     ND             -              -                                     16  10    ND             -              -                                     17  15    0.1            -              -                                     18  0     0.1            -              -                              __________________________________________________________________________     NA = not available                                                            ND = nondetectable                                                            .sup.1 Postvacation samples were drawn after a one month vacation; work 1     samples were drawn six weeks after returning to work; work 2 samples were     drawn more than two months after returning to work                       

The positive results for particular oncogene expression in these workersare of interest, since it is known that foundry workers are at increasedrisk for the development of lung cancer (IARC, 1984, supra). The workerwith the isolated positive result for ras proteins is notable, sincePAHs are known to activate the ras gene (Balmain and Pragnell, 1983,supra; Marshall et al., Nature 310:586 (1984)) and ras gene activationhas been found frequently in human lung cancers, particularly of thenon-small-cell variety (Slamon et al., Science 224:256 (1984); Rodenhuiset al., New Eng. J. Med. 317:929 (1987); Kurzrock et al., Cancer Res.46:1530 (1986)). For example, in a recent study of lung cancer(Rodenhuis et al., 1987, supra), 50% of adenocarcinoma patients (5 of10) had an activated form of one type of ras gene (K-ras and it was feltthat this oncogene activation represented a relatively early event inthe development of the lung cancers and was related to cigarettesmoking. In our own studies of serum oncogene protein levels innon-small-cell lung cancer patients, 15 of 18 patients had increasedexpression of the ras gene products, and 12 of these were known to havebeen cigarette smokers. Increased urinary levels of ras oncogene proteinproducts in lung cancer patients have also been demonstrated (Niman etal., 1985, supra). In our screening of the sera of 16 clinically healthyhazardous waste workers with known carcinogen exposure, one individualhad an extremely abnormal increased expression of ras gene protein,supra. This individual also had the worst history of exposure tocarcinogens including asbestos and cigarette smoke. Thus, it is possiblethat ras gene activation plays a significant role in pulmonarycarcinogenesis, particularly that caused by exposure to environmentalcarcinogens such as PAHs. However, in the current study as noted above,the individual (patient 5) with elevated serum ras proteins was found tobe positive on only one of the three samples. The significance of thisisolated finding in terms of this individual's risk for the developmentof lung or other cancers remains uncertain.

As noted, however, two workers (patients 2 and 7) had consistentlyelevated serum levels for fes oncogene-related protein products. One ofthese workers was a caster in the Zimmerman process with high exposureto PAHs for the past 15 years, smoked cigarettes and had a father andmother who both died of lung cancer. The other worker had been acore-setter with medium exposure to PAHs for the past 9 years. Both ofthese individuals also had occupational dermatitis and had long-termtreatment with topical steroids. Whether topical exposure to steroidsinfluenced the metabolic activation of PAHs or whether the dermatitisincreased dermal absorption of the carcinogens is not clear. Like ras,fes has been found to be frequently expressed in human lung cancers. Inone study, all four lung cancers studied were positive for increasedexpression of the fes gene (Slamon et al., 1984, supra). In our priorstudy of serum oncogene protein levels in lung cancer patients, 11 of 18individuals were positive for fes products, and 8 of these were known tohave been cigarette smokers. Increased urinary levels of fes oncogeneproducts have also been found in lung cancer patients (Niman et al.,1985, supra). In addition, in our study of clinically healthy hazardouswaste workers, only heavy cigarette smokers (6 of 12) were found to bepositive for the fes gene products in their serum. Furthermore, it isknown that during mammalian development, the protein product of the fesgene is expressed in a very limited tissue-specific fashion (Pimentel,Oncogenes. Boca Raton: CRC Press (1986)); for example, in young chicks 6to 18 days old, the fes gene product is detected at high levels only inthree tissues--bone marrow, liver and lung (Mathey-Prevot et al., Cell28:897 (1982)). Thus, in pulmonary carcinogenesis, the expression of thefes gene products may signal a regression to an earlier stage ofpulmonary cellular development, and, once again, this expression may beparticularly associated with exposure to the carcinogens of cigarettesmoke such as PAHs. Therefore, in this study, the next logical step isto determine if the two workers with known long-term medium-to-highlevel workplace exposure to PAHs and known high levels of PAH-DNAadducts and with consistently hgh levels of fes oncogene proteinproducts in their serum are the individuals in this cohort most at riskfor the development of malignant disease, especially cancer of the lung.

Further long-term follow-up of this and other similar cohorts will benecessary before the predictive value of specific oncogene proteins asmarkers for cancer development can be precisely ascertained. However,the current results show that serum oncogene proteins are usefulmolecular epidemiologic markers for the surveillance of populations atrisk for the development of occupational cancers.

While the examples set forth herein specifies the use of monoclonalreceptors, other receptors, such as nucleic acid sequences correspondingto the oncogene-related proteins and related polypeptides hereindiscussed, may be used, and methods for such preparation and use arewithin the skill of the art. In addition, to the extent other bodysamples, such as tissue or urine, may be used for the above assay, thosemethods, described herein, are within the skill of the art.

IX. MATERIALS AND METHODS

A. Growing Of Viruses And Cell Lines

An uninfected mink lung cell line (CCL64), the same line productivelytransformed with the Snyder-Theilen strain of feline sarcoma virus(ST-FeSV) and feline leukemia virus B (FeLV-B) and designated MSTF, aswell as the same line non productively infected with Gardner-Arnsteinfeline sarcoma virus (GA-FeSV) and designated 64F3Cl7 were cultured asdescribed in Sen et al., Proc. Natl. Acad. Sci. USA, 80, 1246-1250(1983). A non-producing avian myeloblast cell line, non-productivelyinfected with avian myeloblastosis virus was cultured as described inDuesberg et al., Proc. Natl. Aced. Sci. USA, 77, 5120-5124 (1980). Thenon-producing marmoset cell line, non-productively infected with simiansarcoma virus (SSV) and designated NPV/SiSV and NPVI/SISV were culturedas described in Devare et al., Proc. Natl. Aced. Sci. USA, 80 731-735(1983). The avian fibroblast non-productivity transformed cell lineinfected with Fujinami sarcoma virus (FSV) was a gift from B. Sefton ofthe Salk Institute, La Jolla, Calif. Uninfected mouse NIH 3T3 fibroblastcells and mouse NIH 3T3 fibroblast cells productively infected withHarvey murine sarcoma virus were cultured as described in Todaro et al.,J. Cell Biol., 17, 299-313 (1963); and Harvey Nature, 204, 1104-1105(1964). Human T24 bladder carcinoma cells were cultured as described inBubenik et al., Int. J. Cancer, 11, 765-773 (1973).

B. Synthesis of Peptides

Polypeptides were synthesized using solid phase methods as described inMarglin and Merrifield, A. Rev. Biochem., 39, 841-866 (1970), and wereconfirmed by amino acid analyses. Sequence information is derived fromeither amino acid sequencing of the viral protein or predictions basedupon nucleotide sequencing. The sources of the sequence information wereas listed in the footnotes relating to those sequences and theironcogenes.

For polypeptides having fewer than 35 residues that were used inimmunizing inocula, a cysteine residue was added to the amino-terminusor to the carboxyl-terminus of each polypeptide whose correspondingoncoprotein sequence did not contain such a residue. The Cys residueswere used to assist in coupling to a protein carrier as described below.

In preparing a useful synthetic polypeptide by the above solid phasemethod, the amino acid residues were linked to a cross-linked resin(solid phase) through an ester linkage from the carboxy-terminalresidue. When the polypeptide was linked to a carrier via a Cys residue,that Cys residue was conveniently used as the carboxy-terminal residuethat was ester-bonded to the resin.

The alpha-amino group of each added amino acid was typically protectedby a tertiary-butoxycarbonyl (t-BOC) group prior to the amino acid beingadded into the growing polypeptide chain. The t-BOC group was thenremoved by standard techniques prior to addition of the next amino acidto the growing polypeptide chain.

Reactive amino acid side chains were also protected during synthesis ofthe polypeptides. Usual side-chain protecting groups were used for theremaining amino acid residues as follows: O-p-(bromobenzyloxycarbonyl)for tyrosine; 0-benzyl for threonine, serine, aspartic acid and glutamicacid; S-methoxybenzyl for cysteine, dinitrophenyl for histidine;2-chlorobenzoxycarbonyl for lysine and tosyl for arginine.

Protected amino acids were recrystallized from appropriate solvents togive single spots by thin layer chromatography. Couplings were typicallycarried out using a ten-fold molar excess of both protected amino acidand dicyclohexyl carbodiimide over the number of milliequivalents ofinitial N-terminal amino acid. A two molar excess of both reagents mayalso be used. For asparagine, an equal molar amount ofN-hydroxy-benzotriazole was added to the protected amino acid anddimethyl formamide was used as the solvent. All coupling reactions weremore than 99% complete by the picric acid test of Gisin, Anal. Chem.Acta. 58:248-249 (1972).

After preparation of a desired polypeptide, a portion of the resulting,protected polypeptide (about 1 gram) was treated with two milliliters ofanisole, and anhydrous hydrogen fluoride, about 20 milliliters, wascondensed into the reaction vessel at dry ice temperature. The resultingmixture was stirred at about 4 degrees C. for about one hour to cleavethe protecting groups and to remove the polypeptide from the resin.After evaporating the hydrogen fluoride at a temperature of 4 degrees C.with a steam of N₂, the residue was extracted with anhydrous diethylether three times to remove the anisole, and the residue was dried invacuo.

The vacuum dried material was extracted with 5% aqueous acetic acid (3times 50 milliliters) to separate the free polypeptide from the resin.The extract-containing solution was lyophilized to provide anunoxidized, synthetic polypeptide from the resin. The extract-containingsolution was lyophilized to provide an unoxidized, syntheticpolypeptide.

C. Coupling of Synthetic Polypeptides To Carrier Protein

The unoxidized synthetic polypeptides were coupled to the carrierprotein keyhole limpet hemocyanin (KLH) through a cysteine residue (Cys;C) of the polypeptide with m-maleimidobenzoyl-N-hydroxysuccinimide esteras the coupling reagent as described in Green et al., Cell, 28, 477 and487 (1982), Where a Cys residue was a terminal residue in a sequence, anadditional cysteine residue was not added.

Briefly, as a generalized procedure for each polypeptide, 4 milligramsof KLH in 0.25 milliliters of 10 millimolar sodium phosphate buffer (pH7.2) were reacted with 0.7 milligrams of MBS that was dissolved indimethyl fermamide (DMF), and the resulting admixture was stirred for 30minutes at room temperature. The MBS solution was added dropwise toensure that the local concentration of DMF was not too high, as KLH isinsoluble at DMF concentrations of about 30% or higher. The reactionproduct, KLH-MB, was passed through a chromatography column preparedwith Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, N.J.)equilibrated with 50 millimolar sodium phosphate buffer (pH 6.0) toremove free MBS. KLH recovery from peak fractions of the column eluate,monitored at 280 nanometers, was estimated to be approximately 80%.

The KLH-MB so prepared was then reacted with 5 milligrams of polypeptidedissolved in 1 milliliter of buffer. The pH value of the resultingreaction composition was adjusted to 7-7.5, and the reaction compositionwas stirred at room temperature for 3 hours.

D. Immunization And Fusion

1. fes-Related Polypeptides

Polypeptides such as those corresponding in amino acid residue sequenceto a portion of the ST-FeSV v-fes oncoprotein were coupled to KLH, andwere used to immunize 129 GIX⁺ mice as described before and in Niman etal., in Monoclonal Antibodies and T Cell Products, Katz ed., (BocaRaton, Fla., CRC Press, Inc., 1982), pp. 21-51. Spleen cells from thoseimmunized mice were fused with SP2/0-Ag14 myeloma cells usingpolyethylene glycol (PEG) 1500 (J. T. Baker Chemco, Phillsburg, N.J.);PEG solutions for fusion were prepared at least one month prior to useto promote fusion efficiency. SP2/0-Ag14 Cells do not produce their ownIg molecules, thereby facilitating isotype analysis and subsequentpurification, such cells also do not produce retroviruses. The fusedcells were then resuspended in 400 milliliters of Dulbecco'shigh-glucose minimal essential medium (Flow Laboratories, Inc.Inglewood, Calif.) containing 10 percent fetal calf serum, 1.0×10-6⁻⁶molar hypoxanthine, 1×10⁶ molar methotrexate, and 1.6×10⁻⁵ molarthymidine. Next, the cells were plated into 30 microliter plates andgrown as described in Niman et al., Proc. Natl. Acad. Sci. U.S.A., 1982supra.

2. sis- and myb-Related Polypeptides

Polypeptides (c) and (d) whose amino acid residues correspond topositions 139-155 of the predicted sequence of simian virus transformingprotein p28^(sis) and to residues 2-18 of the predicted sequence of theavian myeloblastosis virus oncoprotein were synthesized and coupled to aKLH carrier as described above. The conjugates so prepared wereadministered at approximately 50 micrograms of polypeptide per 129 GIX⁺mouse per injection.

On day 0 (zero), each conjugate was mixed with complete Freund'sadjuvant and injected intraperitoneally. On day 19, each conjugate wasadmixed with alum to provide a concentration of 5 milligrams permilliliter of alum, and injected intraperitoneally. A booster injectionof polypeptide (c) in phosphate-buffered saline was administeredintravenously on day 62. Serum containing oligoclonal antibodies wastaken by orbital puncture on day 67. After a second alum-containingimmunization of polypeptide (d) on day 41, the booster of polypeptide(d) was similarly administered on day 143 to similarly provideoligoclonal antibodies on day 148. The serum so obtained was tested forthe antigenicity of its receptors as discussed in FIG. 4.

In a similar manner, polypeptides such as those corresponding to thebelow listed amino acid residue sequences were synthesized.

    ______________________________________                                        abl            LRRACWQWNPSDRPSF                                               fms            FMQACWALEPTRRPTF                                               src            LMCQCWRKDPEERPTF                                                              LGQGCFGEVWMG                                                                  GSSKSKPKDPSQRRRS                                               fgr            AMEQTWRLDPEERPTF                                               ______________________________________                                    

Immunization was carried out in a manner similar to that described forthe sis and myb amino acid residue sequences.

3. ras- and erb B-Related Polypeptides

Polypeptides such as those corresponding in amino acid residue sequenceto residues 96-118 of the ras polypeptide from the predicted sequence ofthe ras oncogene of Kirsten murine sarcoma virus and residues 366-381 ofthe erb B polypeptide from the arian erythroblastemia virus weresynthesized and coupled to a KLH carrier as described above. Theconjugates so prepared were administered at approximately 50 microgramsof polypeptide per 129 GIX⁺ mouse per injection.

On day 0 (zero), each conjugate was mixed with complete Freunds adjuvantand injected intravenously. On day 5, serum containing oligoclonalantibodies was taken by orbital puncture. The serum so obtained wastested for the antigenicity of its receptors as discussed in FIG. 4.

E. Antibody Binding Assay

Hybridomas producing anti-polypeptide antibodies were detected with anenzyme-linked immunoabsorbent assay (ELISA) method as discussed in thedescription of FIG. 4, herein, and in Niman et al., MonoclonalAntibodies and T Cell Products, supra. Briefly, approximately 50micromoles of polypeptide were dried onto microliter plates, fixed withmethanol, and incubated with hybridoma tissue culture supernatant. Afterthorough washing, hydridoma antibody binding was detected using rabbitanti-mouse kappa chain antibody (Litton Bionetics Inc., Kensington, Md.)followed by a glucose oxidase conjugated goat anti-rabbit antisera.Binding was visualized with 2,2'-azino-di3-ethyl-benzothiazoline-sulfonate (6)! (ABTS) dye (Boehringer-Mannheim,Indianapolis, Ind.) in the presence of glucose and horseradishperoxidase as described in Niman et al., Monoclonal Antibodies and TCell Products, supra. Isotype was determined by substituting variousrabbit anti-mouse lambda or heavy chain sera for the anti-mouse kappachain as described above.

F. Electrophoretic Transfer and Immunological Detection of Proteins onNitrocellulose

Cell extracts were subjected to polyacrylamide gel electrophoresis, andthe protein was transferred to nitrocellulose (Schleicher and Schuell,Inc., Keene, N.H.) as discussed in the description of FIG. 5, herein,and in Niman et al., Virology, 123, 187-205 (1982). Peroxidase-labeledrabbit anti-mouse IgG serum (Tago, Inc., Burlingame, Calif.) diluted1/1000 was incubated with the transfers for one hour at 25 degrees C.followed by washing as described in Niman and Elder, in MonoclonalAntibodies and T Cell Produces, above. The bound antibody was visualizedby incubation in 10 millimolar Tris(2-amino-2-(hydroxymethyl)-1,3-propanediol), pH 7.4, 0.009 percent H₂ O₂0.0025 percent 3,3'-dimethoxybenzidine dihydrochloride (Eastman-Kodak,Co., Rochester, N.Y.).

G. Preparation of Purified PDGF

Sixteen units of outdated platelets were obtained from the San DiegoBlood Bank, San Diego, Calif. The purified PDGF used herein was obtainedfollowing the first two steps of the procedures described in Antonaideset al., Proc. Natl. Acad. Sci. USA, 76, 1809-1813 (1979).

Briefly, platelets were collected by centrifugation at 28,000× gravity(g) for 20 minutes at 4 degrees C. The obtained platelets were washed byresuspension in 400 milliliters of a mixture containing (a) 9 volumes of17 millimolar Tris-HCl, at pH 7.4 including 0.15 molar NaCl and 1%glucose; and (b) 1 volume of a solution that includes per 100milliliters: 0.8 grams citric acid monohydrate, 2.2 grams anhydrousdextrose and 2.6 grams of sodium citrate dihydrate, followed by furthercentrifugation at 28,000×g for 10 minutes at 4 degrees C. The thuswashed platelets were then resuspended in 16 milliliters of an aqueoussolution containing 0.008 molar NaCl and 0.01 molar phosphate ion at pH7.4 (NaCl-phosphate ion solution), and boiled for 10 minutes to lyse thecells.

Phenylmethyl sulfonyl fluoride and Traysylol (Sigma Chemical Co., St.Louis, Mo.), protease inhibitors, were added to the lysed cells atconcentrations of 1 millimolar and 3%, respectively. The lysed cellmixture was again centrifuged to provide a pellet and a supernatant.

The supernatant was mixed with 8 milliliters of CM Sephadex C-50(Pharmacia Fine Chemicals, Piscataway, N.J.) beads that were previouslyequilibrated in the NaCl-phosphate ion solution. The beads and liquidwere poured into a chromatography column (15×1.5 centimeters) that waswashed with 6 column volumes of the above NaCl-phosphate ion solution.The PDGF, first eluate, was obtained by eluting the column with twocolumn volumes of 1 molar NaCl. Traysylol was added to the eluate toprovide a final concentration of 3%, and the eluate was dialyzed againstthe above NaCl-phosphate ion solution.

The above-produced lysed cell pellet was extracted with a 1 molar NaClsolution for 24 hours at 4 degrees C., and centrifuged. The supernatantwas dialyzed against the above NaCl-phosphate ion solution, admixed withthe above Sephadex, and made into a column. The column was washed andeluted as above to provide a second eluate that was dialyzed as above.The pellet prepared in this procedure was treated the same way toprovide a third eluate that was again dialyzed as discussed before.

The three dialyzed eluates were pooled and concentrated to a fewmilliliters of volume using an Amicon ultrafiltration apparatus (Amicon,Lexington, Mass.) and a filter having a 10K dalton exclusion. The PDGFso purified was then treated as discussed for FIG. 5.

Purified PDGF extract from approximately 2.5 units of platelets weremixed with a minimal volume of solution containing 0.5 percent sodiumdodecyl sulfate (SDS) and 5 percent of 2-mercaptoethanol. The resultingmixture was boiled for two minutes and then electrophoresed therethrougha 5-17 percent polyacrylamide gel. The protein was thereafterelectrophoretically transferred to nitrocellulose. (Niman and Elder,supra.) that was thereafter cut into strips, following the Western blotprocedure.

The nitrocellulose strips so prepared were then treated with a solutioncontaining 3 percent bovine serum albumin (BSA), 0.1 percentpolyoxyethylene-9-octyl phenyl ether (Triton® X-100) in phosphatebuffered saline to inhibit non-specific protein binding. Fourmilliliters of mouse anti-serum diluted 1:200 were then incubated withthe nitrocellulose strips.

After washing three times with a solution of 0.1 percent Triton® X-100in PBS, the nitrocellulose strips were incubated either with 10⁶ countsper minute of ₁₂₅ I-labeled Staphylococcus aureus protein, or a 1:1000dilution of peroxidase-conjugated goat anti-mouse serum (Tago), andagain washed with 0.1 percent Triton® X-100 in PBS. The peroxidaseconjugate was developed with a solution containing 0.0009 percent H₂₀ 2,0.0025 percent 3,3'-dimethoxybenzidine dihydrochloride (Eastman-Kodak,Co.,) in a 10 millimolar Tris buffer having a pH value of 7.4. The ¹²⁵ Ilabeled strips were developed by exposure on XRP-1 film (Eastman-KodakCo.) using Cronex Hi-Plus (E.I. DuPont de Nemours & Co.) intensifyingscreens at minus 70° C. for 48 hours.

H. Urine Assay

Urine from donors (patients) as noted in the description of the Figureswas collected and used as collected or concentrated to 40-fold using anAmicon ultrafiltration apparatus. This fluid was employed as the bodyfluid sample aliquot in the assay for proteins encoded by or related tosis, fes and ras oncogenes.

The concentrated urine sample was prepared in the following manner. Theurine was clarified at 6000 r.p.m. at 4° C. for 10 minutes. Thesupernatant was then concentrated using an Amicon filter having a 10,000dalton exclusion. This concentrated urine was then dialyzed to separateprotein fractions.

Concentrated urine was electrophoresed at 25 microliters per lane into a5-17% polyacrylamide gel to provide the equivalent of protein from oneml of collected urine, and then electrophoresed onto nitrocellulose. Thenitrocellulose filter was then probed with a 1/200 dilution of, forexample, mouse antiserum in a solution 3% bovine serum albumin, 0.1%Triton® X-100 and PBS. The nitrocellulose filter was then washed threetimes and incubated with 10⁶ cpm of ¹²⁵ I-labeled protein A.

Binding was visualized with intensifying screens at -70° Centigrade asdescribed in FIG. 6, supra.

I. Oncoproteins and Transformed Cells

NRK and SSV-transformed NRK cells were provided by S. A. Aaronson and K.C. Robbins of the Center for Cancer Research, National Institutes ofHealth, Bethesda, Md. The cells were grown in Dulbecco's minimalessential medium supplemented with 10% fetal calf serum, 2 millimolarL-glutamine, 100 IU per milliliter of penicillin and 100 micrograms permilliliter of streptomycin.

Parallel cultures of NRK and SSV-transformed NRK cells were washed 3times for 2 hours intervals, and were then incubated for 18 hours inmedium without serum at 15 milliliters per T75 centimeter² flask. Themedium so conditioned was then centrifuged, and was stored frozen at-70° C.

The conditioned medium was thawed, concentrated 500-fold using dialysisin i molar acetic acid and was thereafter lyophilized. Aftersolubilization and reduction with 10% 2-mercaptoethanol, 50 microlitersof concentrated, conditioned media were electrophoresed into a 5-17%sodium dodecyl sulfate polyacrylamide gel. Secreted proteins were thenelectrophoretically transferred and bound to nitrocellulose. Nonspecificbinding was blocked by preincubation of the cell extract with a solutioncontaining 3% of bovine serum albumin and 0.1% polyoxyethylene octylphenyl ether in phosphate buffered saline at a pH value of 7.4.

Prior to carrying out the immunological assays, 20 microliters of mouseantisera induced by PDGF-2(1-18) or PDGF-2(73-89) (described before)were preincubated with 100 micrograms of an appropriate polypeptide for1 hours at 37° C. The oligoclonal antibody-containing/polypeptidereaction mixture was then diluted 1:500 with the above preincubationsolution. The diluted solution so prepared was then contacted at 4° C.with the nitrocellulose-bound conditioned media, and that contact wasmaintained (incubated) for a time period of 15 minutes, a timesufficient for the immunoreaction of the antibody (receptor) and proteinbound on the nitrocellulose. The nitrocellulose was thereafter washed.

The washed nitrocellulose was then contacted with affinity-purifiedrabbit anti-mouse IgG₁ antibodies (Litton) diluted 1:500 at 25° C. Thecontact was maintained for a time period of 2 hours sufficient for theanti-mouse IgG₁ antibodies to immunoreact with antibodies from theantisera that had bound to the nitrocellulose-bound secreted proteins ofthe conditioned media. The nitrocellulose was then washed again.

Immunoreaction (binding) was visualized with 10₆ counts per minute of125I-labeled Staphylococcus aureus protein A as described in Niman,Nature, 307, 180-183 (1984).

J. Oncoproteins in the Urine Samples of Newborns and Pregnant Mothers

The monoclonal receptors utilized were prepared as described previously.One ml of urine from each of the newborns was admixed with sufficient2-mercaptoethanol to make a 10 volume percent solution. The resultingsolution was boiled for 2 minutes. Upon cooling, aliquots of theresulting reduced solution were electrophoresed on 5-17 percentpolyacrylamide gels. The proteins of the resulting gels were transferredto nitrocellulose following standard procedures. Nitrocellulose blotsfor each urine sample were individually screened for immunoreactivitywith each of the antibody probes following standard procedures for suchWestern blots. Autoradiography was for 4 hours at -70° C. using Cronexintensifying screens. The relative intensities of immunoreaction werethereafter determined.

Urine samples from pregnant (expectant) mothers were concentrated priorto electrophoresis. Here, proteins from serial urine collections takenin a time period 16-20 weeks into the pregnancy (based upon the lastmenstrual cycle) were first precipitated from the urine samples byadmixture with 2 volumes (based on the urine volume) of acetone andmaintenance at 4° C. The precipitated proteins were collected andresuspended using 1/20 of the original sample volume of PBS. The sex ofthe fetuses being carried was determined either by amniocentesis orvisual inspection after birth.

The United States Government has rights in this invention pursuant toPublic Health Service Contract NOl-CP-41009, Public Health ServiceGrants CA 38160 and CA25803.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the novel conceptsof the invention.

We claim:
 1. A method of assaying a body sample from a host for anindicant of exposure to a carcinogen prior to clinical symptoms ofcancer, comprising the steps of:(a) contacting said body sample with areceptor molecule, wherein said receptor binds to both (i) anonco-protein to form an onco-protein/receptor complex and ii) a peptidehaving an amino acid residue sequence containing about 7 to about 40amino acids corresponding to a portion of said onco-protein, and, (b)determining the level of said onco-protein in said body sample bydetecting the amount, if any, of said onco-protein/receptor complex;wherein an elevated level of said onco-protein, relative to said levelin a normal sample, is indicative of exposure of said host to at leastone carcinogen.
 2. A method of claim 1 wherein said body sample isselected from the group consisting of blood, tissue, and urine.
 3. Amethod of claim 1 wherein said assay is an immunoblot.
 4. A method ofclaim 1 wherein said receptor molecule is selected from the groupconsisting of a polyclonal antibody, an oligoclonal antibody, amonoclonal antibody and a fragment thereof.
 5. A method of claim 1wherein said receptor molecule binds to both (a) H-ras oncogene-relatedp21 and (b) a polypeptide having an amino acid residue sequence, fromleft to right and in the direction, from amino terminus to carboxyterminus represented by a formula selected from the group consisting of:##STR3##
 6. A method of assaying a body sample from a host for anindicant of exposure to a carcinogen prior to clinical symptoms ofcancer, comprising the steps of:(a) contacting said body sample with areceptor molecule, wherein said receptor binds to both (i) H-rasoncogene-related p21 protein to form a p21 protein/receptor complex, and(ii) a peptide having an amino acid residue sequence, from left to rightand in the direction from amino terminus to carboxy terminus,represented by a formula selected from the group consisting of: ##STR4##and, (b) determining the level of said p21 protein by detecting theamount, if any, of said p21 protein/receptor complex; wherein anelevated level of said p21 protein, relative to said level in a normalsample, is indicative of exposure of said host to at least onecarcinogen.